Selection, breeding and engineering

of microalgae for bioenergy and

biofuel production
Anthony W.D. Larkum
1
, Ian L. Ross
2
, Olaf Kruse
3
and Ben Hankamer
2
1
Climate Change Cluster, University of Technology (Sydney), Broadway, NSW 2007, Australia
2
Division of Chemistry and Structural Biology, The Institute for Molecular Bioscience, 306 Carmody Road, The University of
Queensland, Saint Lucia, Brisbane, Qld 4072, Australia
3
Center for Biotechnology (CeBiTec), Department of Biology/Algae Biotechnology and Bioenergy, Bielefeld University, 33615
Bielefeld, Germany
Microalgal production technologies are seen as increas-
ingly attractive for bioenergy production to improve fuel
security and reduce CO
2
emissions. Photosynthetically
derived fuels are a renewable, potentially carbon-neutral
and scalable alternative reserve. Microalgae have partic-
ular promise because they can be produced on non-
arable land and utilize saline and wastewater streams.
Furthermore, emerging microalgal technologies can be
used to produce a range of products such as biofuels,
protein-rich animal feeds, chemical feedstocks (e.g. bio-
plastic precursors) and higher-value products. This
review focuses on the selection, breeding and engineer-
ing of microalgae for improved biomass and biofuel
conversion efciencies.
Global signicance of microalgae for solar energy
harvesting
Photosynthetic organisms including higher plants, micro-
algae and cyanobacteria fulll the crucial role of capturing
solar energy and storing it as chemical energy. Annually,
5500 ZJ (550010
21
J) of solar energy reaches the Earths
atmosphere [1]. This corresponds to 12 000 times the
annual global energy demand (0.46 ZJ) [2] and illustrates
that solar energy is not limiting for signicant scale up of
microalgal biotechnologies for the production of food, fuel,
chemical feedstocks and high-value products. The amount
of solar energy currently captured by arable crops is limit-
ed by arable land area (3.9% of the Earths surface area),
fresh water (1% of global water), nutrient supply and
solar energy-to-biomass conversion efciency (ecosystems
<1%; optimized crop production; 0.52%) [35]. Advan-
tages of microalgal biotechnologies include the possibility
of increased photosynthetic efciency, location on non-
arable land (25% of the Earths surface) and the use of
saline and wastewater sources. Short life cycles also allow
near continuous harvesting. Although arable crops can in
theory achieve similar areal efciencies, in practice they
are less likely to do so because they are affected by varia-
tions in soil quality, water availability, nutrient supply and
cropping seasonality.
In parallel with biofuel systems [6,7], articial photo-
synthesis systems are also being developed. Recent analy-
sis of articial photosynthetic systems capable of
producing fuel (e.g. photochemical panels used to drive
water photolysis to generate H
2
) and microalgae indicated
that the former could achieve higher efciencies [8]. How-
ever, these calculations excluded construction energy, rare
earth metal availability and life cycle analysis. Inclusion of
construction energy, which in the case of microalgae
includes basal metabolism, brings the relative efciency
of microalgae and articial photosynthesis processes clos-
er, as will microalgal cultivation systems that optimally
use incident light and minimize the energy required for
construction and used for mixing [9]. Perhaps more impor-
tantly, however, articial photochemical biofuel systems
can currently only produce H
2
, whereas microalgae can
produce a wide range of carbon-based foods, fuels and
chemical feedstocks of higher value. The importance of
this is that market adoption of a technology is likely to
be driven by economics ($/carbon atom xed) rather than
absolute efciency (photons/carbon atom xed). In the
context of developing bioenergy systems with improved
efciency and product diversity, microalgal systems there-
fore have considerable potential.
The ability to site microalgal systems on non-arable
land and to use saline and wastewater is an increasingly
important consideration for policy makers as the global
population, with its need for food, fuel, chemical feedstocks
and fresh water, is forecast to increase from 7 billion to 9
billion by 2050 [10]. In the face of an estimated 30%
increase in water demand by 2050 [11] and the depletion
of essential nutrients such as phosphate [12,13], the use of
closed-loop microalgal culture systems will be of vital
importance [9]. A crucial question therefore is whether
microalgal systems can be economic. First-generation
microalgal production facilities have focused on the com-
mercial production of high-value products such as b-caro-
tene (Dunaliella spp.) and health food supplements
(Spirulina), nutraceuticals and sh feeds [3,14]. Despite
initial setbacks [15,16] a transition to viable microalgal
biofuel systems, perhaps via co-production models,
appears to becoming a more realistic goal as technological
Review
Corresponding author: Larkum, A.W.D. (a.larkum@sydney.edu.au).
198 0167-7799/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.11.003 Trends in Biotechnology, April 2012, Vol. 30, No. 4
developments advance, co-product prices (e.g. animal feed)
rise and international carbon trading schemes are intro-
duced [17]. However the development of this technology is
in its early stages and much remains to be done at the level
of optimizing the biology, engineering, business models
and life cycle analysis, as well as in testing pilot- and
demonstration-scale systems. This article specically fo-
cuses on optimization of the biology component through the
search for microalgae species and the selection of strains
and their subsequent genetic modication to rene sus-
tainable fuel production systems [4].
Microalgae and biofuel production
Microalgal diversity
Large-scale production of microalgae has the potential to
match the biomass productivity gains that were achieved
through the agricultural revolution on which our modern
society has become dependent. However, the green revolu-
tion of the 20
th
century occurred over a matter of decades,
and to realize similar gains, large-scale selection and
breeding programs for microalgae (and cyanobacteria) will
also be required.
Microalgae are a diverse group within the protists, the
simplest form of eukaryotic life (Box 1, Figure 1). The
microalgal species that form the main genetic base for
biofuel research and production (Table 1) represent a very
Box 1. Microalgae and cyanobacteria
Microalgae are small (110 mm) photosynthetic eukaryotic organ-
isms. Technically, microalgae do not include the cyanobacteria,
which are prokaryotic organisms. However, cyanobacteria in fact
gave rise to the chloroplasts of eukaryotic algae (and land plants)
and they share many features, such as the ability to drive
photosynthetic water photolysis, and thereby contribute to the
production of both atmospheric oxygen and reduced organic
carbon. Today, cyanobacteria also have much promise for bio-
energy production and therefore are included in this article. True
algae are taxonomically located at the base of the tree of eukaryote
organization and form part of the diverse group called protists
(Figure 1). Some of the best-known algae are multicellular (macro-
algae such as seaweeds) and include the green, brown and red
algae. The majority of the worlds algae are, however, unicellular.
Because of their ancient lineage (1.52 billion years old) and
greater genetic diversity than land plants (which form one super-
class of plants, tracheophytes or land plants; Figure 1), algae form
many classes spread throughout the protists and offer very wide
diversity (Figure 1). This huge gene pool and limited morphological
scope make it difficult to answer the question of how many
microalgae there are, and we currently have insufficient data to
answer this question. Conservative estimates give a figure of 30 000
algal species. However, this estimate only includes macroalgae
(seaweeds) and does not include phytoplankton, the free-floating
microalgae of lakes and seas. Brodie and Zuccarello estimated that
there are 350 000 algal species in total by extrapolating from large
and species-rich taxa [18]. In all probability, the actual figure is much
higher than this. Regardless of how many there are, it is clear that
algae (including Cyanobacteria here in the broadest sense) have
adapted to a wide range of conditions that include saline, freshwater
and terrestrial environments, hot and cold springs, a great range of
mineral compositions, very low to very high light conditions, and
many other environmental factors. Microalgae therefore provide us
with an enormous resource of genetic variation and chemical
diversity.
Melawimonas californiana
Melawimonas jacobiformis
Trichomonas
vaginalis
Giardia
lamblia
Jacobids
Excavata
Euglenozoa
Red algae
Green algae
Higher plants
Glaucophytes
Haptophytes
Cryptophytes
Stramenopiles
Ciliates
C
h
r
o
m
o
p
h
y
t
e
s
Dinoflagellates
Apicomplexans
Rhizaria
Reclimonas americana
Jacoba libera
Euglena gracilis
Porphyra yezoensis
Galdieria sulphuraria
Cyanidioschyzon mer.
Arabidopsis thaliana
Zea mays
Oryza sativa
Glaucocystis nostochinearum
Cyanophora paradoxa
Pavlova lutheri
Emiliana huxleyi
Isochrysis galbana
Goniomonas pacifica
Guillardia theta
Thalassiosira pseud
Chlamydomonas reinhardtii
Leishmannia major
Trypanosoma cruzi
Trypanosoma brucei
Phytophthora sojae
Phytophthora ramorum
Tetrahymena therm.
Paramecium tetra.
Karenia brevis
Alexandrium tamar.
Toxoplasma gondii
Bigelowiella natans
1
2
Melawimonas californiana
Melawimonas jacobiformis
Trichomonas
vagi ggg nalis
Giardiaa
lamblia
Jacobids
Excavata
Euglenozoa
Red algae
Green algae
Higher plants
Glaucophytes
Haptophytes
Cryptophytes
Stramenopiles
Ciliates
Dinoflagellates
Apicomplexans
Rhizaria
Reclimonas americana
Jacoba libera
Euglena gracilis
Porphyra yezoensis
Galdieria sulphuraria
Cyanidioschyzon mer.
Arabidopsis thaliana
Zea mays
Oryza sativa
Glaucocystis nostochinearum
Cyanophora paradoxa
Pavlova lutheri
Emiliana huxleyi
Isochrysis galbana
Goniomonas pacifica
Guillardia theta
Thalassiosira pseudd
Chlamydomonas reinhardtii
Leishmannia major
Trypanosoma cruzi
Tryp TTTTTTTTT anosoma brucei
Phytophthora sojae
Phytophthora ramorum
Tetrahymena therm.
Paramecium tetra...
y
Karenia brevis
Alexandrium tamar.
Toxoplasma gondii
Bigelowiella natans
1
2
Reticulomyxa filosa
TRENDS in Biotechnology
Figure 1. Phylogenetic tree showing the placement of algae and higher plants
among some of their common protist neighbors. Colored lines and letters indicate
photosynthetic branches and groups. Black lines and groups indicate non-
photosynthetic branches and groups. The land or higher plants (tracheophytes)
evolved from the green algal branch. The algal branch [1] to green algae contains
most of the species used in biofuel research. The algal branch [2] to
Eustigmatophytes, in the Haptophyte group, contains two species,
Nannochloropsis oculata and Pavlova lutheri, used in biofuel research. Tree
redrawn, with permission, from [71].
Table 1. Microalgae commonly used for bioenergy and the organic chemicals industry
Algal species Algal class Product(s) Culture technique Advantages or drawbacks Refs
Botryococcus braunii Chlorophyceae Triterpene oils Photobioreactor Whole genome available [63]
Chlorella spp Chlorophyceae Carbohydrates, protein Ponds,
photobioreactor
Widespread and adaptable [64]
Chlamydomonas
reinhardtii
Chlorophyceae Oils, carbohydrates,
hydrogen and methane
Photobioreactor Transformable [28]
Dunaliella salina Chlorophyceae b-Carotene Brackish seawater
ponds
Needs high salinity [65]
Nannochloropsis Eustigmatophyceae Polyunsaturated
fatty acids
Seawater ponds Lipid bodies produced under
nitrogen stress
[6]
Ostreococcus tauri Prasinophyceae
(Chlorophyceae)
Oils Photobioreactor Smallest known microalga,
whole genome available
[66]
Pavlova lutheri Prymnesiophyceae Fatty acids, aquaculture
feedstock
Photobioreactor Little used [67]
Arthrospira platensis
(Spirulina)
Cyanobacteria Health food Ponds,
photobioreactor
Filamentous morphology [68]
Synechocystis and
Synecococcus
Cyanobacteria Isoprenes, oils Photobioreactor Whole genomes available,
transformable
[69]
Review Trends in Biotechnology April 2012, Vol. 30, No. 4
199
small fraction of the vast and largely untapped biodiversity;
their selection is still mainly based on ad hoc choices. For a
small subset of these, mutagenesis, genetic engineering and
synthetic biology approaches are being developed to
improve product yields and production-related qualities
(e.g. product type, ease of harvesting, disease resistance).
Microalgal strains used for biofuel production
The rst stage in increasing the genetic base for the
breeding of algae for biofuel production is to explore their
vast natural biodiversity, which is estimated to consist of
approximately 350 000 species (Box 1) [18]. To date, pub-
lished biofuel studies have focused on less than 20 species
taken from culture collections (Figure 2) and of these
publications, 70% are not comparative studies, referring
to only one of the genera in Figure 2. The workhorses for
process development for microalgae and cyanobacteria
have been Chlorella and Synechocystis. Pioneering groups
focusing on the development of novel biofuel systems have
identied a wider range of promising genera shown in
Table 1. Of these genera, nearly all comprise a single test
species. Although these strains have been selected from a
larger pool of species such as the 3000 strains collected
during the Aquatic Species Program [19], the literature on
algal biofuels reveals little exploration of the wider algal
biodiversity. One practical reason is that it is easiest to use
strains that have been well documented. Perhaps a deeper
reason is a perception that there is little to be gained in
terms of biomass productivity by examining new strains,
because the ultimate limits to biomass production in a
bioreactor are set, not by the strain, but by external con-
straints including light inux, photosynthetic and meta-
bolic limits and by the intrinsic times needed for cellular
replication [20]. In this view, under maximal growth con-
ditions, commonly used biofuel strains already grow at or
near the limits of the energy impinging on the system. If
particular strains have advantages for biofuel production,
these advantages must reside in other properties such as
the accumulation of desired storage compounds (e.g. high-
lipid strains), high-value products (e.g. unsaturated fatty
acids), salt and temperature tolerance, resistance to pre-
dation and ease of harvesting. There are, however, many
other properties that should be improved and which would
benet from a broad sampling of natures genetic diversity,
such as the ability to grow at high cell density and the
overexpression and excretion of specic products.
The concept of nding or engineering new high-lipid-
producing strains is attractive because triacylglycerides
can be converted simply and easily to biodiesel (fatty acid
methyl esters) via a rapid and technically straightforward
process [21]. Consequently, much algal biofuel research
has focused on the development of high lipid yield [22]. It is
often noted that some species (e.g. Nannochloropsis spp.
and Botryococcus braunii) can accumulate up to 80% of
their dry cell mass as intracellular lipid. However, high
yields per cell (which may take a long time to accumulate)
are not as important as the total system biofuel yield
(measured as yield m
2
day
1
) or indeed their cost of
production. Solar irradiation (as a rst approximation)
depends on surface area rather than volume, so areal
productivity (biomass g
1
m
2
day
1
) is important for photo-
autotrophic systems and a crucial variable for economic
analysis [3,17].
Microalgal hydrogen production has been the subject of
much research [2325], as have other biofuels such as
methane [26], ethanol [27] and dimethyl furan [28]. For
these fuels, low-lipid algal biomass can also be exploited.
Consequently, low or moderate lipid-producing strains,
which efciently store chemical energy as protein or car-
bohydrate, may also be viable biofuel sources given a
(a) (b) 900
800
700
600
500
400
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Hydrogen
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Lipids
TRENDS in Biotechnology
Figure 2. Algal strain-specific publications since 1991. (a) The Web of Science was searched for algae and biofuels and publications were subsequently divided according to
species. The references presented capture 70%of all algal biofuel publications. (b) Publication number by year for algal biofuel publications referring to biodiesel, hydrogen
and lipids.
Review
Trends in Biotechnology April 2012, Vol. 30, No. 4
200
suitable downstream process and market conditions (e.g.
higher fuel prices in Europe than in the USA). The use of
co-production models (e.g. oil- and protein-rich animal feed
or the co-production of chemical feedstocks such as bio-
plastic precursors) and high-value products (including re-
combinant products) can be used to develop economic
viability [17].
A variety of biomass-to-liquid fuel conversion strategies
are also being developed [2931]. These are designed to
utilize the entire biomass and not just the lipid fraction.
Such methods simplify downstream processing and include
the use of hydrothermal liquefaction to convert wet algal
biomass [32]. This approach eliminates the need to use
energy to dry the algal biomass, and thus improves the
energy balance of the process. Downstream fractionation of
algal oil also opens up the opportunity to target premium-
priced biofuels such as jet fuel. Regardless of the fuel type
of interest, economic analyses suggest that utilization of all
biomass components can improve economic viability [17].
The selection of a wide variety of traits is therefore clearly
desirable, because this diversity will allow the best species
and processes to be matched.
Stepping stones to the microalgal future
Of the less than 20 strains developed for biofuel production
(for representatives of these, see Table 1), few are well
developed for genetic manipulation. Consequently, most
improvements were achieved by manipulating the bio-
chemistry of the cultures to produce, for example, more
lipid [21,3335]. Nannochloropsis oculata, a eustigmato-
phyte alga placed in the Haptophyte group (Figure 1), like
other biofuel algae, naturally produces storage lipid glo-
bules. Biochemical manipulation has been used to improve
lipid production in Nannochloropsis through nitrogen star-
vation and CO
2
enrichment [33]. However, there is limited
literature reporting on the possibilities of transformation
in Nannochloropsis (using Agrobacterium methods; [36]).
The only microalga that reportedly can be genetically
transformed with any degree of efciency at present is
Chlamydomonas reinhardtii (Figure 1). This green alga
does not gure highly in microalgal bioenergy production,
except in the area of hydrogen production, as it is not
widely reported to concentrate lipid reserves. However,
its genetic tractability could change this in the near future
[37] and in any case lessons learnt from C. reinhardtii will
no doubt be transferred to other species.
Type culture collections versus ad hoc collections
Type culture collections are based on extensive experience
in collecting, characterizing and maintaining strains in an
axenic state over long periods of time and have the advan-
tage that the risks of strain loss, contamination, misiden-
tication and genetic drift are minimized [38]. However,
there are also many advantages to collecting local strains.
First, many algal strains in collections have been propa-
gated by continuous culture under specic selection con-
ditions over long periods, rather than being cryopreserved
[38,39]. Continuous subculture results in adaptation to the
selection conditions and the accumulation of mutations
that can compromise the robustness or phenotypes of
the original wild-type organisms and their performance
in commercial production systems. The local collection of
strains and rapid cryopreservation can eliminate this ge-
netic drift and preserve traits related to adaptation to the
local climatic conditions, herbivores and pathogens. Sec-
ond, in certain countries, quarantine restrictions add a
signicant layer of regulatory difculty. Third, although
many algal culture collections are currently free of intel-
lectual property (IP) restrictions, this may change in the
new biofuels era. Culture collections are likely to come
under increasing pressure to assert IP rights over the
strains they hold. Paradoxically, this will make such IP-
constrained collections less interesting and valuable be-
cause much contemporary algal research is driven by
industry focused on securing proprietary IP. Furthermore,
given the enormous biodiversity of algae and their ease of
access, the resource in question is not a scarce one and the
barriers to entry (i.e. collection from the wild) are not likely
to be high. The real IP value in algal strains will probably
lie in engineered or mutant strains for which an IP license
represents an easier and cheaper route to obtaining the
desired capability. Given the rapid expansion of algal
biotechnologies, existing IP protection strategies through
alternative models are being developed [40] and may be
more benecial to rapid advancement of the industry than
traditional structures.
To reduce the time required to collect and isolate new
algae strains, automated processes are being developed
[41,42], for example through the use of ow cytometry and
robotics (Box 2). Sophisticated cryopreservation techni-
ques and new descriptive technologies such as deep se-
quencing and microuidics [4347] also offer the potential
to dramatically increase the rate of isolation, preservation,
identication and characterization of algal species at the
molecular level. In parallel, metagenomic approaches are
documenting the extraordinary diversity of microbial life
in natural ecosystems and have demonstrated that current
stocks of culturable organisms represent a minority of
species actually present in the environment (Box 1). It is
likely, therefore, that among this diversity and ecological
richness, there are algae and algal populations that pos-
sess traits that (i) we currently desire and (ii) of which we
are currently ignorant but could use if they were discov-
ered. This suggests that despite the natural possibility of
multinational agribusinesses dominating the eld, the
opportunity for smaller-scale innovative enterprises will
continue for many years to come.
Breeding strategies and photobioreactors
Microalgae have signicant advantages over land plants
for breeding. First, they have life cycles of hours or days
instead of seasonal cycles. Second, their largely unicellular
nature greatly assists in the miniaturization of breeding
systems, which reduces cost. Third, eukaryotic algae (but
not cyanobacteria) are able to replicate both sexually and
asexually, which accelerates the generation of genetic
diversity compared to prokaryotic organisms. Fourth,
microalgae can be selected and screened for particular
phenotypes using ow cytometry or other high-throughput
methodologies (Box 2). Fifth, UV and chemical mutagene-
sis can be more easily applied to microalgae than higher
plants and the application of specic selection pressures
Review Trends in Biotechnology April 2012, Vol. 30, No. 4
201
(see adaptive evolution below) can greatly enhance the rate
of strain development.
The need for analyses of microalgal growth parameters
under controlledconditions (light intensity, pH, CO
2
supply,
mixing) that can be deployed with speed and reliability has
spawned the development of many types of photobioreactor
(at plate, tubular, helical and other more complex geomet-
ric designs); these are rapidly expanding in the diversity of
their design and may make a major contribution to future
algal bioenergy production solutions [9]. Although the ulti-
mate eld trials will be in large-scale installations such as
ponds, raceways andexisting closed photobioreactors, many
of the above designs can be tested at a small scale.
Molecular strategies
Adaptive evolution is essential for species survival and
reproduction. However, this does not mean that species are
genetically programmed for optimized mass production of
a particular product under large-scale operational condi-
tions. Thus, many key parameters require careful improve-
ment based on both genetically and non-genetically
manipulated organisms (GMOs and non-GMOs).
Transgenic microalgae are a focus of growing interest,
with an opportunity to construct new and highly efcient
phenotypes [48,49]. New strains can be developed by spe-
cically targeting genes using reverse or forward genetics
strategies. In the latter approach, screening for random
knockout libraries has much to offer. However, for most
algae being developed for biofuel production, the lack of
transformation techniques is currently a major limitation.
An alternative strategy is the isolation and breeding of
highly efcient non-GMO strains. Such an approach can
involve high-throughput screening of libraries after chem-
ical treatment or UV mutagenesis, which avoids the regu-
latory problems of using GMO strains in outdoor
production systems. With automated screening techni-
ques, this approach is becoming very attractive. Successful
attempts with the haploid yeast Pichia stipitis [50] based
on transcriptome analysis combined with backcrossing
approaches will open up opportunities to probe microalgal
mutants with high bioenergy production capacity in non-
GMO strains. Nevertheless, this approach still requires
molecular biology techniques based on a haploid, fully
sequenced and annotated genome.
Box 2. Selection criteria for bioenergy production
Microalgae can be purified froma wide range of environments using a
variety of techniques, including physical extraction from crude water
samples (e.g. micromanipulation) as shown in Figure I, dilution to
resolve individual cells, antibiotic selection and enrichment cultures
using specific selection pressures (e.g. photoautotrophic conditions).
Individual algal strains can be purified based on traditional colony
selection or high-throughput fluorescence-activated cell sorting (FACS)
approaches before the resulting axenic cultures are cryopreserved for
storage to prevent genetic drift. Screening of algae is often based on
optimal growth or the production of specific metabolites and the use of
response surface modeling and principle component analysis to
identify these conditions and the key variables controlling them.
50m
50m
50m
50m
50m
Water
samples
Antibiotic
selection
Algae
enrichment
Cell sorting
Optimum
growth
Response
surface
Metabolite
profiling
Principal
component analysis
Cryo
storage
Crude
water
Dilution Selection
plates
b c
TRENDS in Biotechnology
Figure I. Microalgae purification and screening.
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Construction and application of engineered microalgal
mutants
Successful selection, construction and molecular analysis
of the genotypes of GMO and non-GMO mutant strains
require comprehensive knowledge of the microalgal ge-
nome and access to molecular and gene manipulation tools,
including selectable markers, vectors and techniques for
systematic insertion in screening libraries. Advanced tar-
geted gene up- and downregulation approaches and other
facets of transcriptional regulation, such as RNAi, will also
benet this research [51]. The important role of non-coding
RNAs and their effect on the regulation of metabolite ow
opens up another important eld of study [52]. Currently
there are only a few microalgae species that fulll even
some of these crucial requirements. Apart from Chlamy-
domonas reinhardtii and Phaeodactylum tricornutum [53],
only Chlorella kessleri [54], Porphyridium [55] and fairly
recently Nannochloropsis [56] and Dunaliella salina [57]
have been successfully transformed, but as genomes and
the factors regulating them are increasingly understood,
new tools will be developed.
For successful transformation of microalgae, hurdles
associated with gene efciency, integration or stability
need to be overcome. This will require suitable vectors
for successful incorporation, reporter genes and suitable
homologous promoters. New DNA incorporation methods
(e.g. optimized homologous recombination strategies) and
databases for modeling species-specic codon usage be-
tween donor and host organism would also be benecial,
as would be a wide selection of fully sequenced species with
mononucleated cells and separate sexes for future genetic
crossing [48].
C. reinhardtii remains the model organism of choice
owing to its sequenced genome and extensive tool set [58].
In addition, a random insertion library has been created
that enables researchers to identify mutants with gene
defects of interest directly via PCR analysis with gene-
specic primers by screening DNA superpools. Reverse
genetics approaches are important new tools for direct
isolation of mutants of interest; this overcomes the problem
whereby attempts to produce targeted knockouts of nuclear
encoded genes are typically prevented by the inability of C.
reinhardtii to perform efcient homologous gene recombi-
nation in the nucleus. In the future it should be possible to
combine these genetic approaches and incorporate key
changes to produce mutants with desirable properties.
Although Chlamydomonas is furthest developed, this
situation is rapidly changing and many patents are in the
pipeline for species including Nannochloropsis and
Dunaliella, although published papers remain scarce [36].
Blue sky possibilities
One of the major challenges for microalgal biofuel produc-
tion is maximization of the light capture efciency of photo-
synthesis, because this is the rst step in all biofuel
production processes. Of the 43% of the energy in the solar
spectrum that can be capturedvia photosynthesis, wild-type
strains can only convert approximately 48% to chemical
energy in the form of biomass [4,8]. Table 2 highlights the
reasons for the difference between the 43% incident and 4
8% converted energy and identies theoretical solutions to
improve photosynthesis efciency. Areas of particular inter-
est include the development of photosynthetic systems with
coordinated pigment sets to yield expanded absorption and
excitation ranges of photosynthetically active reaction cen-
ters in both the green and red regions of the spectrum. The
recent discovery of chlorophyll f [59], which absorbs in the
near-infrared region, illustrates the potential of surveying
biodiversity and suggests that coordinated insertion of a
range of pigments (e.g. chlorophyll a, b and d) could yield
such an expanded absorption range. In concert with an
expanded absorption range, a reduction in total antenna
size may also have promise, because this approach yields
improved photosynthetic efciency due to lower losses of
captured energy through non-photochemical quenching
[6062]. Furthermore, because photosynthesis stores solar
energy via the chemical reduction of CO
2
, the complex
interplay between spectral range, light capture efciency
andCO
2
xation by rubisco is a crucial areaof research. This
is because the rate of photosynthesis is limited by the
activity of rubisco, which catalyzes the competing reactions
of CO
2
xation and oxygenation (photorespiration). Balanc-
ing of CO
2
and O
2
supply to rubisco while maximizing the
light reactions is a complex process involving both biology
and engineering.
Another area of fundamental research is the develop-
ment of models of regulatory networks in algae to assist in
better gene and metabolic regulation to, for example,
optimize the storage of chemical energy in a particular
form (e.g. a specic oil). In addition, an understanding of
Table 2. Photosynthesis efciency problems and solutions
Problem Theoretical solution
Chlorophyll works as a quantum device and only the energy
of the rst excited singlet state is used in energy conversion;
40% of the visible spectrum is unavailable for photosynthesis
Extend the absorption range of the photosynthetically active pigments
in the wavelength range capable of driving photosynthesis (<700 nm)
a
Energy losses for non-photochemical quenching under
light-saturated conditions
Increase the rate of CO
2
xation and thereby reduce photoinhibition
Microalgae use two photosystems in series, resulting in
further inefciencies
Introduce articial photosystems such as a single system capable of
splitting water and producing NADPH; photophosphorylation would
have to be adjusted to supply sufcient ATP for carbon xation
Energy losses due to basal metabolism and cell division; this can
however be seen as the internalized energy cost of producing the
system, just as solar panels have an internalized energy cost of
production (Box 2)
Develop fuel production systems (e.g. bio-H2) that are more closely
coupled to the electron transport chain than the synthesis of oils and
are thus subject to lower energetic losses
a
Pigments that absorb in the yellowand green region of the spectrumor phycobiliproteins can be introduced to extend the absorption range. They have already been used
to solve this problem(e.g. red algae, cryptophytes and prokaryotic cyanobacteria). In recent years we have also seen the discovery of two newchlorophylls, d and f [59,70].
Review Trends in Biotechnology April 2012, Vol. 30, No. 4
203
the signaling mechanisms of algal cells in more complex
algal populations and the development of pathogen and
predator resistance will allow more precise biological con-
trol of future production systems.
Any of these discoveries could advance the eld from the
development of systems producing fuels for the existing
liquid fuel market, such as those required by the aviation
industry, through to completely new types of fuel for the
future, such as cleaner burning dimethyl ether and hydro-
gen. There is great potential to be tapped, given that only a
minute fraction of algal species have been isolated, char-
acterized, sequenced and developed for genetic manipula-
tion, selection or breeding. However, meaningful progress
in all of the above areas and the many others that could be
mentioned will require a great deal of work and, inevitably,
time. However, as we become increasingly constrained in
terms of fossil fuel supplies, food and water, the pressure
for innovative changes to microalgal production is only
likely to intensify.
Acknowledgments
We are grateful to the Australian Research Council (A.W.D.L., I.R. and
B.H.) for funding that supported this research. B.H also gratefully
acknowledges the support of the Queensland Government. O.K. gratefully
acknowledges the DFG (KR1586/4, KR1586/5), the European Commission
(EU-FP7 contracts 212508 and 245070) and the German Federal Ministry
of Science (BMBF contracts 0315265A, 03SF0361G) for funding.
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