Available online at www.sciencedirect.

com

Potential of industrial biotechnology with cyanobacteria and

eukaryotic microalgae
René H Wijffels1, Olaf Kruse2 and Klaas J Hellingwerf3

Both cyanobacteria and eukaryotic microalgae are promising higher plants. Both prokaryotic cyanobacteria (commonly
organisms for sustainable production of bulk products such as referred to as blue-green algae) and eukaryotic microalgae
food, feed, materials, chemicals and fuels. In this review we will are commercially promising producers of chemicals and
summarize the potential and current biotechnological biofuels.
developments.Cyanobacteria are promising host organisms for
the production of small molecules that can be secreted such Commercial large-scale cultures of the eukaryotic micro-
as ethanol, butanol, fatty acids and other organic acids. alga Chlorella were started in the early 1960s followed by
Eukaryotic microalgae are interesting for products for which the cyanobacterium Arthrospira in the 1970s. By 1980
cellular storage is important such as proteins, lipids, starch large-scale algae production facilities were established in
and alkanes.For the development of new and promising lines Asia, India, the USA, Israel and Australia. Food supple-
of production, strains of both cyanobacteria and eukaryotic ments from microalgae comprise an important market in
microalgae have to be improved. Transformation systems which compounds such as b-carotene, astaxanthin, poly-
have been much better developed in cyanobacteria. unsaturated fatty acid (PUFA) such as DHA and EPA and
However, several products would be preferably produced
polysaccharides such as beta-glucan dominate [1,2].
with eukaryotic microalgae. In the case of cyanobacteria a
synthetic-systems biology approach has a great potential to
Research on algae is not only focusing on improving pro-
exploit cyanobacteria as cell factories. For eukaryotic
duction of traditional products but also on new algae pro-
microalgae transformation systems need to be further
ducts such as biodiesel, bio-ethanol and chemicals [3,4,5].
developed. A promising strategy is transformation of
Microalgae are considered as one of the most promising
heterologous (prokaryotic and eukaryotic) genes in
feedstock for biofuels and chemicals. Worldwide, research
established eukaryotic hosts such as Chlamydomonas
and demonstration programs are being carried out to
reinhardtii.Experimental outdoor pilots under containment
develop the technology needed to expand algal energy
for the production of genetically modified cyanobacteria
and chemicals production from a craft to a major industrial
and microalgae are in progress. For full scale production
process [6–10]. Apart from the boost in algal research
risks of release of genetically modified organisms need to
generated by the oil crisis and environmental motives
be assessed.
(climate change, CO2 issues and land use) also technical
Addresses developments such as the introduction of new generation
1
Wageningen University, Bioprocess Engineering, AlgaePARC, P.O. Box
8129, Wageningen 6700 EV, The Netherlands
DNA sequencers, improvements in algal genetic modifi-
2
Bielefeld University, Dep. of Biology, Center for Biotechnology cation, and the rise of systems biology have contributed to
(CeBiTec), Bielefeld 33615, Germany the expanding of research on algae. Metabolic pathways can
3
University of Amsterdam, Laboratory for Microbiology, Swammerdam be introduced, deleted or changed [9,11,12].
Institute for Life Sciences, P.O. Box 94232, Amsterdam 1090 GE, The
Netherlands
Benefits of using both eukaryotic microalgae and cyano-
Corresponding author: Wijffels, René H (Rene.Wijffels@wur.nl) bacteria for sustainable production of fuels and chemicals
will be summarized. We will indicate that despite being
promising still a large number of bottlenecks need to be
Current Opinion in Biotechnology 2013, 24:405–413
removed before eukaryotic microalgae and cyanobacteria
This review comes from a themed issue on Energy biotechnology become industrial microorganisms.
Edited by Eric Toone and Han de Winde
For a complete overview see the Issue and the Editorial One of the items often mentioned is the use of dedicated,
Available online 4th May 2013
improved strains. In this paper we will focus on that and we
will identify which features need to be improved. Further-
0958-1669/$ – see front matter, Published by Elsevier Ltd.
more we will focus on dedicated, improved strains and
http://dx.doi.org/10.1016/j.copbio.2013.04.004 identify which further features need to be improved, for
both cyanobacteria and eukaryotic microalgae. Transform-
ation systems are much better developed in cyanobacteria
Introduction but some products would be better to produce in eukar-
Algae are generally defined as photosynthetic microor- yotic microalgae, particularly when high intracellular pro-
ganisms. They are considered as simple organisms duct storage capacity is required. Finally aspects of scale up
because they are not organized into organs found in will be discussed.

www.sciencedirect.com Current Opinion in Biotechnology 2013, 24:405–413

406 Energy biotechnology

Products neutral site [22,23], as well as extra-chromosomally via

A number of biofuels can be produced from eukaryotic conjugation with replicable plasmids. For each organism
microalgae and cyanobacteria. The whole biomass can be several resistance markers and random mutagenesis pro-
digested into methane or processed into crude oil via cedures are available, and markers can be deleted after
pyrolysis and other thermo-chemical processes. The dis- mutagenesis. Basic information about weak, strong and
advantage of using the whole biomass for energy only is inducible promoters is available, as well as detailed infor-
that not all the value of the biomass is used. Difficulty mation about regulatory sequences, needed for trans-
may reside in cost effectively producing biofuels, whereas lation. Care should be taken, after modification of the
added value compounds promise to be important drivers chromosome, to check proper segregation, as these cya-
for microalgae development. By biorefinery it is possible nobacteria are polyploidic [24], although it significantly
to refine the biomass into different valuable products extends the time required for such modifications.
[5,13]. Quite some efforts have been done to produce
hydrogen gas, both from eukaryotic microalgae and cya- With these methods, various metabolic pathways have
nobacteria. However, this route is in a very early stage of been introduced into Synechocystis and Synechococcus, which
development [14]. We believe that for biofuel purposes then allow the recombinant strains to produce any of a
especially production of carbohydrates (cellulosis and series of products such as: ethanol [25,16], lactic acid
starch), which can be converted into ethanol and butanol, [26,27], sucrose [28], ethylene [29], isoprene [30],
direct production of ethanol and butanol in cyanobacteria iso-butyraldehyde [31], iso-butanol [32], and 1-butanol
and production of lipids which can be converted into [33] (see Figure 1). For lactic acid and sucrose production
biodiesel are the most promising routes [7,8,10,15,16]. a gene encoding a solute transporter was included in the
cloned metabolic pathway [26,28] to release the product
Like for transport fuels, organic chemicals and polymers in the extracellular medium. For other products such as
are based on fossil oil. The primary compounds are lactic acid [27] and butanediol (O Borirak, unpublished
dominated by a small number of key building blocks, information) introduction of a such a transporter is not
which are mainly converted to polymers and plastics. required. Initially product levels in the exracellualer
Eukaryotic microalgae produce hydrocarbons and poly- medium were in the mg/l range, but in 2012 for several
saccharides which can be converted into building blocks products g/l concentration were achieved (e.g. [34,35]).
such as ethylene, propylene, adipic acid and furanics [17– For some products this implies the need for the appli-
20] (www.eu-splash.eu). Some of these building blocks cation of product removal (because of product toxicity,
can also be directly produced by genetically engineered see e.g. [35]), whereas for other, more water soluble
microalgae. products such as sucrose and lactic acid, these toxicity
effects only occur at (sub)molar concentrations. Gener-
Products we would like to focus on are: ally, product toxicity increases with the octanol/water
partitioning, but with a complex, non-linear, relation
- from cyanobacteria: ethanol, butanol, fatty acids and (Table 1). Productivity of the best producing strains is
other organic acids and accumulation of storage still between 1 and 2 orders of magnitude below the rate
compounds (carbohydrates) of CO2 fixation that the cells can maximally achieve.
- from microalgae: proteins, lipids, starch, alkanes and
from that the different chemicals and fuels that can be After expansion of cyanobacterial metabolism with a
derived heterologous metabolic pathway, and optimization of
the pathway itself [33], further optimization of pro-
duct-formation and product-level can be (and has been)
Cyanobacteria achieved by streamlining the cyanobacterial CO2-fixation
Research towards application of cyanobacteria for the and intermediary metabolism. For the production of
production of biofuel and/or bulk chemicals has been sucrose this is not even necessary, as a simple salt stress
focused on Synechocystis and Synechococcus, with much makes the cells channel nearly all (i.e. up to 90%) its fixed
smaller additional efforts being carried out in Anabena CO2 into this sugar (Ducat et al., 2012), which leads these
and Cyanothece (a nitrogen-fixing strain). Representative authors to conclude that ‘Sucrose production in engin-
strains from all four groups/genera have been sequenced, eered S. elongatus compares favorably with sugarcane and
but for Synechocystis this information is available already other agricultural crops’. However, most of the above
since 1997 [21]. This has been followed up by an exten- mentioned products are derived from pyruvate. The level
sive systematic description of the organism, including of pyruvate metabolite in wild type Synechocystis is rather
application of nearly all ‘omics’ techniques. In both low [36]. To increase product formation in that case
Synechocystis and Synechococcus all basic molecular-genetic pathways that catabolize pyruvate need to be eliminated
techniques for metabolic engineering are available. and pyruvate synthesis pathways need to be re-enforced.
Heterologous genes can be introduced into their genome, This has been demonstrated amongst others for the
via natural transformation and so-called docking at a production of ethanol, lactic acid, etc. The usefulness

Current Opinion in Biotechnology 2013, 24:405–413 www.sciencedirect.com

 Industrial biotechnology cyanobacteria and microalgae Wijffels, Kruse and Hellingwerf 407

Figure 1

CO2

RI1,5BP 3PG
ATP ATP

Calvin
Sucrose Benson
RI5P Cycle 1,3BPG

PII ATP ATP ADP GAP

NADPH
ADP-Glc G1P GIc G6P F6P FDP
glgC Isoprene
3PGA
1-Butanol
Glycogen
Amylose Asparate and phaA
Cyanophycin PEP PYR ACCoA PHB storage
storage
CO2
Glycogen CO2
cphA PDC LDH ALS NADP+
storage
MAL OA IIvC
Acetaldehyde Lactate Acetolactate DHIV

ADH ALDc CO2 IIvD

FUM CIT

TCA Pathway Ethanol Acetoin Ketoisolaverate

AR KDc
SUCC ICIT
Butanediol Isobutyraldehyde
SUCC ADH
αKG Ethylene
CoA
EFE Isobutanol
Current Opinion in Biotechnology

Schematic overview of intermediary photoautotrophic metabolism of Synechocystis PCC6803, with indication of energy products and feedstock
products that can be derived from these cells.

of this latter type of optimization extends all the way to analyses. For future work one can really expect important
the initial reaction of the Calvin cycle [31], be it that contributions from synthetic systems biology, ones it has
‘sink regulation’ by itself already leads to increased become possible to derive engineering suggestions in
Rubisco activity, provided that the capacity of the intro- synthetic biology from kinetic versions of flux-balance
duced heterologous metabolic pathway is high enough analysis methods. This approach may then help resolve
[27] (Ducat et al., 2012). the involvement of all the complicated mechanisms that
lead to source and sink regulation in photosynthesis in
These latter results clearly show that a synthetic-systems cyanobacteria. Eventually, this may lead to a situation in
biology approach will be beneficial to further exploit which carbon fixed by these ‘cell factories’ is only
cyanobacteria as ‘cell factories’ for the production of diverted to the synthesis of new cells, to the extent that
biofuel and bulk chemicals. Up to now, most of the equals the death rate of the producing cells. Metabolic
synthetic biology work is based on straight-forward meta- control theory (or: a ‘sensitivity analysis’) then provides
bolic pathway information as this has become available for the tool to predict the optimal level of expression of the
organisms such as Escherichia coli and Saccharomyces cere- heterologous product-forming metabolic pathway, to pre-
visiae, whereas systems biology studies have mainly vent this pathway to become a metabolic burden to the
focused on the derivation and application of flux-balance cyanobacterium [27].

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408 Energy biotechnology

Table 1 In addition, eukaryotic microalgae are able to either

Toxicity of various small molecules towards photoautotrophic directly synthesize (e.g. in the chloroplast) or temporarily
growth of Synechocystis PCC6803 in batch culture at low light store (e.g. in the vacuole) products within cellular com-
intensities. Growth rate without additions equals 0.06 hourS1 partments, which clearly increases the flexibility and
Solvent IC50 (mM) Water partitioning Range efficiency of design-driven synthetic biology strategies
coefficient (Log Po/w) tested (mM) by for example offering the opportunity to even produce
Ethanol 868 0.4 0–1000 components that are toxic to the cell. Further advantages
Lactate 382 0.79 0–1000 include easy harvesting due to its large cell size
Acetoin 95 0.66 0–200 (>10 mm). Only a few microalgae including the green
Meso-BD 376 0.59 0–500
algae Chlamydomonas reinhardtii, Nannochloropsis gaditana
2-Butanol 48 0.66 0–100
Acetaldehyde 37.5 0.03 0–50 and Ostreococcus tauri or the diatom Phaeodactylum tricor-
nutum are currently established for genetic engineering
approaches [39,42,43]. For these limited number of
species the necessary molecular tool boxes are available.
Following this strategy, cyanobacteria will be the candi- In particular C. reinhardtii has already been used to con-
date organisms of choice for application as cell factories struct new and highly efficient phenotypes of interest
for small molecules, for which cellular storage capacity is (e.g. [44,45]).
not crucial, but (energetic) efficiency in biosynthesis is.
Eukaryotic microalgae in contrast are to be preferred for Based on the comparable easy way to perform heter-
those applications in which (transient) cellular storage is ologous gene insertion in C. reinhardtii, this microalga
important. successfully expresses edible therapeutics in the chlor-
oplast [46–48].

Eukaryotic microalgae C. reinhardtii is reported as generally safe with a very

The availability of an increasing variety of unicellular limited risk of toxic contamination by bacteria or viruses.
eukaryotic microalgae for genetic manipulation are the Apart from chloroplast expression, nuclear gene expres-
reason that these microorganisms are nowadays widely sion combined with cytosolic protein production is also
used for genetic and metabolic engineering approaches established to a certain extent in this model organism, but
aiming for the production of both proteins and secondary the efficiency still needs to be improved by codon optim-
metabolites. The eukaryotic phenotype shows certain ization of the nuclear genome and by introduction of
advantages for applications in the field of molecular suitable promoter sequences.
biotechnology, with many of them already described in
established bio-economy driven approaches over the last For (heterologous) protein expression, it is known that
decade [37,38]. eukaryotic systems are in particular preferred for pro-
duction processes, when post-translational modification
Unicellular eukaryotic microalgae combine required (PTM) such as glycosylation is required [49,50]. The
and preferred features of both, plant systems and bac- success of functional protein production often relies on
terial systems such as fast growth, rapid transformation glycosylation for correct protein folding, for maintaining
cycles, scalability, capacity for photo-mixotrophic protein activity and for achieving an appropriate expres-
growth and low cultivation costs. The genome of many sion rate. A lack of such eukaryotic post-translational
eukaryotic microalgae consists of a mixture of bacterial- modification capabilities requires complicated processing
type, plant-type and animal-type genes [39]. As typical steps for the efficient purification of proteins.
protists, these organisms are therefore often referred as
multifunctional ‘planimals’ [40]. Because of their In addition to protein production, eukaryotic algae pro-
specific and complex genetic background eukaryotic duce several secondary metabolites of interest for bio-
microalgae have retained a variety of metabolic path- technological purposes. Some eukaryotic microalgae
ways making them attractive for biotechnologically naturally contain high levels of triacyglcerols with distinct
driven approaches. Many species can switch between fatty acid profiles making them an attractive source for the
phototrophic growth and heterotrophic growth under petrochemical industry. In the background of a huge
aerobic conditions and are able to perform anaerobic number of so far uncharacterized algae species world-
fermentation. Recently, it was also shown that eukar- wide, the search for algae with interesting neutral lipid
yotic microalgae can use cellulose as an alternative profiles will continue [51,13]. At the same time, new
carbon source for growth [41], a phenotype which advanced strategies are going to be developed to enhance
has been so far only described for non-photosynthetic lipid production in eukaryotic microalgae [52,53,54,55].
organisms. This very interesting phenotype opens up The lipid metabolism in microalgae is very complex and
new perspectives for using cellulosic waste material for not yet fully understood [56] making it difficult to identify
algae cultivation. targets for efficient molecular engineering approaches.

Current Opinion in Biotechnology 2013, 24:405–413 www.sciencedirect.com

 Industrial biotechnology cyanobacteria and microalgae Wijffels, Kruse and Hellingwerf 409

New molecular techniques for the synthesis of hydro- Figure 2

carbons by a solar-driven production process will come to
the fore, since efficient synthesis of for example, short- (a) (b)
chain and long-chain alkanes has the potential to provide
a sustainable and renewable source for high-value com-
modity chemicals. In order to achieve this goal with
eukaryotic microalgae, it is however essential to overcome
current limitations avoiding an energy and time-consum-
ing product extraction from biomass by developing and
designing direct catalytic sun-to-fuel pathways in which
photon energy and atmospheric CO2 is directly converted (c) (d) (e)
into carbon-based products with sufficient photon con-
version efficiencies. Advanced synthetic biology strat-
egies are already used to re-design microalgae for
catalytically driven direct synthesis of low and high-value
products [57–59]. In this context, systematic analyses of
the efficient triterpene and alkane production pathways
in Botryococcus braunii [60] could serve as a blue print for
heterologous genetic engineering approaches in other
eukaryotic microalgae.

The efficient production of non-volatile components in

eukaryotic microalgae also requires fast secretion mech-
anisms (Figure 2). A separation of the product from the
catalytic reaction site reduces the risk of protein degra-
dation by cytosolic proteases, avoids product feedback
inhibition and even offers the opportunity to produce
and accumulate components that have the potential of
negative interference with the cell metabolism. Efficient
product secretion is therefore also a crucial pre-condition
for continuous cultivation strategies. The process of pro-
duct secretion is well understood for proteins in eukaryotic Current Opinion in Biotechnology
algae. Protein secretion signals in C. reinhardtii are there-
fore already introduced in state-of-the-art molecular engin- Value of eukaryotic secretion in microalgal recombinant protein
eering strategies. As a proof of concept, the combination of expression. (a,b) Colony screening via bioluminescence signal on petri
synthetic biology, plate level protein abundance assay and plate level for robust expression mutants. (c) Chlamydomonas reinhardtii
harboring vector pcCAgLuc [62] expressing and secreting the Gaussia
recombinant protein quantification was recently used to
princeps luciferase. (d) Culture after centrifugation, clear media is visible.
demonstrate efficient nuclear gene expression combined (e) Addition of 0.01 mM coelenterazine directly to culture media elicits
with extracellular localization in two independent bioluminescence activity. (Figure provided by Kyle J. Lauersen, Bielefeld
approaches [61,62]. In contrast, strategies for engineering University).
efficient hydrocarbon secretion mechanisms into suitable
eukaryotic hosts have not been sufficiently described and
are therefore one of the most challenging tasks in the near Large-scale production of genetically
future of algal biotechnology research. modified eukaryotic microalgae and
cyanobacteria
In summary, the ability of eukaryotic microalgae to Genetic modification of eukaryotic microalgae and cya-
accumulate and store secondary metabolites in cellular nobacteria are now mainly studied in the laboratory. For
compartments and to efficiently produce and secrete industrial production the process should be scaled up and
functionally active protein products make these organ- applied outdoors.
isms ideal hosts and powerful alternatives to cyanobac-
teria in industrial biotechnology. Promising strategies for The production cost in photobioreactors is an important
the construction of more efficient production strains in topic. In many studies attention is given to reduction of
the future will be based on the availability of advanced cost price of production. Production costs are an order of
synthetic biology tools for metabolic pathway engineering magnitude too high for production of commodities from
via transformation of heterologous (prokaryotic and microalgae and cyanobacteria We believe it will be
eukaryotic) genes in established eukaryotic hosts such possible to reduce production costs 10 times if the tech-
as C. reinhardtii. nology develops and if biomass is refined into different

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410 Energy biotechnology

Figure 3

(a) (b)

(c) (d)

Current Opinion in Biotechnology

Algae production systems at AlgaePARC, Wageningen UR, the Netherlands (www.AlgaePARC.com).

products [5]. We refer to the literature on this topic [63] modified organisms and these organisms probably are
and will focus here on risk aspects of culturing genetically poor in competition at conditions occurring in nature.
modified microalgae outdoors. In addition it is suggested for future experiments to
culture the genetically modified strains under extreme
For production in containment organisms can be cultured conditions (salinity, pH, temperature) as a selective pres-
and processed in such a way that protective measures are sure.
used to prevent their direct contact with the environment.
For industrial production outdoors it will be impossible to Until this moment there is still limited practice of algae
work under containment. The escape of genetically production systems. As an example we will give the
modified algae into the environment even from closed regulative procedure we followed for the production
photobioreactors will be unavoidable. It is expected that facility AlgaePARC (Figure 3). Although at AlgaePARC
most of the genetically modified microalgae cultured in we do not work with genetically modified algae we did
production systems have very little chance to survive if follow the procedure for contained use because of the lack
released to the environment. However, it is suggested to of practice of evaluations. Official regulations to use
analyze the associated risks before starting large-scale genetically modified organisms are actually not in place
production [64]. Risk assessments should be focused at AlgaePARC. The measures taken should pave the way
on the chance that organisms released from the cultiva- for such regulations. The following protective measures
tion system would be able to outcompete wild strains. It is were taken at AlgaePARC:
unlikely that genetically modified algae and cyanobac-
teria will outcompete wild strains as the cultivation con- - The closed cultivation systems are situated in a
ditions are specifically designed for the genetically concrete bin with sufficient capacity to hold the content

Current Opinion in Biotechnology 2013, 24:405–413 www.sciencedirect.com

 Industrial biotechnology cyanobacteria and microalgae Wijffels, Kruse and Hellingwerf 411

of the cultivation systems even during heavy rainfall. Acknowledgements

This concrete bin has a drain which automatically closes K.J.H. would like to thank Drs S. Andreas Angermayr for his help in
in case of leakage of a cultivating system; preparing his part of this review.
- The open pond is secured against overflow by means of
a level detector. At maximum water level a pump will References and recommended reading
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the level does not sink within 5–10 min the drain of the
concrete bin will close automatically;  of special interest
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