Renewable energy systems

Algae, a New Biomass Resource 1

Algae, a New Biomass Resource needed for growth. In oxygenic photosynthesis,
light-induced redox reactions occurring in the pho-
CINZIA FORMIGHIERI, ROBERTO BASSI tosynthetic electron transport chain are coupled to
`
Dipartimento di Biotecnologie, Universita di Verona, the extraction of electrons from water.
Verona, Italy Photosystem A multipigment-protein complex com-
posed of a light-harvesting antenna moiety energet-
Article Outline ically connected to a reaction center where the
excitation energy is used for charge separation and
Glossary
production of a reduced product.
Definition of the Subject
Light saturation constant The intensity of light at
Introduction
which photosynthetic oxygen evolution and specific
Advantages of Algae as Biomass Producers
biomass growth rate are half the maximum level.
Present Algal Productivity in the Laboratory Versus
The energy absorbed in excess with respect to the
Large Scale
photosynthesis saturation is dissipated as fluores-
Domestication
cence or heat and not used for photochemistry.
Light Use Efficiency
Photoinhibition The light-induced inactivation of
Non-Photochemical Energy Quenching at the Molecular
photosynthesis occurring when photooxidative dam-
Level
age of the photosynthetic machinery (particularly
Interconversion and Storage of Photosynthetic Metabolic
photosystem II) overcomes the capacity for repair.
Products
Genetic improvement All procedures, including phe-
Screening for State Transition as an Indirect Mean to
notypic selection, conventional breeding, mutagen-
Select Strains with Altered Redox Metabolism
esis, and genetic engineering, aimed at indirectly or
Accumulation of Biomass as Neutral Lipids
directly influencing the genetic background of
Planning an Algal Refinery
a wild strain, which was evolved following rules of
Large-Scale Systems
natural selection. Genetic improvement is intended
Future Perspectives and Technological Developments
at improving existing characteristics or at introduc-
Bibliography
ing new traits to fit applications.
Genetic engineering All the techniques of recombi-
Glossary
nant DNA to directly manipulate genotypes.
Algae Oxygenic photosynthetic organisms, prokary-
otic or eukaryotic, with organization ranging from
Definition of the Subject
unicellular to multicellular. Algae never have true
stems, roots, and leaves, thus leading to their clas- Algae are oxygenic photoautotrophs, offering a very
sification as “lower” plants. high level of biodiversity and thus suitable for different
Biofuel Renewable energy-rich compound derived practical applications. Today, they are mainly cultivated
from living organisms or from their metabolic for human/animal food or to extract high-value
by-products. chemicals and pharmaceuticals. However, their exploi-
Biomass Organic raw material, stored as a result of the tation could be extended. Algae are attractive as high
metabolism of a living organism, which can be used yield biomass producers, because of the short life cycle,
as a resource for energy and biofuels. the ability to grow up to very high cell densities, and the
Photoautotrophy The ability of a living organism to easy large-scale cultivation that does not compete with
use carbon dioxide as carbon source for biomass other demands such as those of conventional crops
and light as source of energy. agriculture. Algae can be a resource of renewable, sus-
Photosynthesis The overall process that converts light tainable biofuels. In addition, they can be transformed
energy into chemical energy, finally used to fix into “cell factories” to produce recombinant proteins of
inorganic carbon dioxide into organic compounds interest for pharmaceutical companies.

M. Kaltschmitt et al. (eds.), Renewable Energy Systems, DOI 10.1007/978-1-4614-5820-3,
# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3

2 Algae, a New Biomass Resource

Introduction a major problem for human society. Its becoming
increasingly clear that finding alternative, renewable
Algae are described as “lower” plants that never have
energy sources to sustain our lifestyle is striking and
true stems, roots, and leaves, and grow photoautotro-
urgent. Such widespread and environmentally sustain-
phically by performing oxygenic photosynthesis [1].
able energy source will allow single countries to be
They are mostly eukaryotic, although prokaryotic
more independent from the escalating prize of crude
cyanobacteria are included in algae. More than
oil. The public conscience that search for energy
200,000 species are estimated and form a highly het-
sources alternative to fossil fuels is fundamental for
erogeneous group, spread in all aquatic ecosystems
future prospects is generally increasing and research is
but also in other habitats such as soil. A broad spec-
undertaken in many countries. Advances have been
trum of phenotypes and specialized adaptation abilities
made recently on the exploitation of renewable energies
exists within this group while members colonize
such as sun and wind, which can be considered as
diverse ecological habitats, from freshwater to marine
“clean” and non exhaustible. Fuels derived from living
and hyper-saline, at different temperatures, pH,
organisms or from their metabolic by-product are
and nutrient availabilities [1, 2]. Algae are classified
defined as “biofuels.” Photosynthetic organisms con-
as follows: cyanobacteria (Cyanophyceae), green algae
vert carbon dioxide into biomass, a form of stored
(Chlorophyceae), diatoms (Bacillariophyceae), yellow-
chemical energy that can be used in substitution for
green algae (Xanthophyceae), golden algae
fossil fuels. In some species of algae, 30% up to 80% of
(Chrysophyceae), red algae (Rhodophyceae), brown
the biomass is accumulated as oil that can be extracted
algae (Phaeophyceae), dinoflagellates (Dinophyceae),
and converted to biodiesel [2, 8–11]. Some algae,
and “pico-plankton” (Prasinophyceae and Eustigma-
mainly green, can photobiologically generate hydrogen
tophyceae). Algae are accountable for the net primary
[12–15], which is released from the culture with
production of $50% of the total organic carbon pro-
a purity up to 98% [16]. Spent algal biomass can be
duced on earth each year [3], a reason for their massive
further valorized to produce biomethane from anaero-
biological importance. Long before the advent of bio-
bic digestion of the biomass itself [1, 5] and high-value
technology, algae were used as a human food source for
ingredients for food and pharmaceuticals can be
centuries by indigenous populations [4, 5]. However,
produced along with biofuels in order to reach cost-
aside from some attempts to cultivate algae and to
effectiveness and economic competitivity over conven-
extract from them valuable products back to the
tional fuels.
seventeenth century [6], commercial large-scale pro-
duction of algae began few decades ago [4, 7]. Cur-
Advantages of Algae as Biomass Producers
rently, algae are mainly cultivated as human/animal
food source and used in aquaculture, in agriculture, The use of algae for fuel production has notable
as fertilizers, and in order to produce high-value advantages. First of all, algae are photoautotrophic
chemicals, pharmaceuticals, and cosmetics, such as organisms, thus able to produce biomass from solar
polyunsaturated o3 fatty acids, proteins, biopolymers, energy, water, and carbon dioxide, all renewable and
and polysaccharides as agar, carrageenan, alginates, cheap components. Carbon dioxide removal, in addi-
pigments, vitamins, and antioxidants [1, 4]. tion, would contribute to reduce atmospheric levels of
Recently, a new interest in algal biomass as renew- this pollutant which, instead, is stoichiometrically
able energy source is emerging. About 80% of world released in the atmosphere by conventional fuels. The
energy demands are met by the combustion of fossil use of fossil fuels currently releases about 6 billion tons
fuels: coal, oil, and natural gas. However, fossil fuel of carbon dioxide per year, only partially compensated
reserves are limited and their exhaustion is estimated, by re-assimilation by photosynthetic organisms in for-
assuming a stable consumption, to occur within few ests (about 1 billion ton yearÀ1) and oceans (2 billion
decades while their combustion has high impact on tons yearÀ1) [17] leading to a 3-billion-ton yearÀ1
environment, due to releases of large amounts of car- increase in the global level of carbon dioxide in the
bon dioxide and other pollutants. This is considered atmosphere. This has been widely suggested to cause


the so-called greenhouse effect, leading to world global 5. Algae can utilize nutrients from a variety of waste-
warming [18]. To prevent undesired climate changes, water sources (agricultural runoff, and industrial
carbon dioxide emissions need to be reduced in the near and municipal wastewaters), providing additional
future [19]. Biofuels from algae are attractive because benefit of bioremediation.
their combustion would not result in net carbon dioxide 6. The life cycle of algae is more rapid (hours to days)
emissions since biofuels will only release the amount of as compared to land plants (months to years),
carbon previously absorbed during growth of algae. allowing for a faster turnover and a higher biomass
Advantages of algae over crop plants are many: yield. In more detail, crop plant life cycle has
a long initial development phase using energy
1. Photosynthesis increases with carbon dioxide con- stored in seeds for building the organism, followed
centration until saturation. Algae can grow with up by a relatively short phase of full photosynthetic
to 18% of carbon dioxide, being more efficient car- activity and then senescence, limiting the period
bon dioxide assimilators than land plants [20] and of efficient light harvesting and utilization to a
suitable for mitigation of carbon dioxide emissions fraction of the year. In contrast, algae not only
when coupled to an industrial activity. Chlorella have a short life cycle but they remain productive
vulgaris was shown to respond well to elevated throughout the year, allowing for a continuous bio-
carbon dioxide levels (over 1,850 ppm CO2) and mass production when light is available and tem-
to effectively remove up to 74% of the carbon perature allows.
dioxide in the airstream to ambient levels (330 7. Many algal species are competent for using
ppm CO2) with a 63.9 g mÀ3 hÀ1 bulk removal in organic carbon as energy source. This ability
the experimental photobioreactor of 2 L [21]. The opens the possibility to allow for biomass pro-
carbon dioxide fixation rate by algal biomass is esti- duction at night by supplying low-priced carbon
mated ten times as large as that of the temperate substrates.
forest [22]. 8. Algal biomass is fully photosynthetically active. In
2. Cultivation of algae does not compete with other contrast, photosynthesis is localized exclusively in
demands such as food production because algae can leafs of vascular plants, a fraction of the total plant
grow in wastelands rather than in arable lands. body, while the rest is an energy sink, thus a disad-
Using the entire US soybean crop for biodiesel vantage in terms of biomass productivity.
would replace only 10% of conventional diesel con-
sumed and total world plant oil production would
Present Algal Productivity in the Laboratory
only satisfy 80% of US demand [23]. Exploiting
Versus Large Scale
algae rather than crops thus represents an essential,
sustainable alternative to fulfill the energetic Algae can convert solar energy into chemical energy
demands without affecting food supply. through the process of photosynthesis. The whole pro-
3. Algae, differently from crops, do not require pesti- cess of oxygenic photosynthesis can be summarized by
cides, which avoids contamination of water and Eq. 1.
soils and also fertilization is limited to the culture
6H2 O þ 6CO2 ! C6 H12 O6 þ 6O2 ð1Þ
vessel without dispersion of nutrients in the envi-
ronment and consequent eutrophysation of water Light energy is used to extract electrons from water
bodies. (H2O), thus generating oxygen (O2). These electrons
4. Algae can grow in seawater, brackish water, or are transported through a linear electron transfer
wastewater making a substantial saving of fresh- chain and finally reduce NADP+ to NADPH. Photo-
water that in contrast is required by conventional synthetic electron transport is coupled to the genera-
crops in high amounts. Extensive cultivation of tion of a transmembrane electrochemical gradient,
algae would therefore be more environmentally whose stored energy is used to synthesize ATP.
sustainable than extensive cultivation of conven- NADPH and ATP are then used to produce glyceralde-
tional crops. hyde-3-phosphate from CO2 in a metabolic pathway


that is called the Calvin–Benson cycle [24]. Photosyn- would be of 77 g biomass mÀ2 dayÀ1 (280 t haÀ1
thesis enables the cell to convert inorganic CO2 into yearÀ1), corresponding to a solar-to-biomass conver-
organic carbons and finally to accumulate biomass. sion efficiency of 8–10% [25]. This value is about 25
A theoretical estimation of algal biomass yield can times higher than estimations for vascular plants, pos-
be assessed considering the efficiency of photosynthe- sibly due to algal biomass being fully photosyntheti-
sis. A minimum of 8 photons of light energy are cally active. Such estimation assumes that all available
absorbed per each oxygen molecule evolved, the actual light energy is absorbed and utilized for photosynthe-
average measurement is 9.5 photons per oxygen [25]. sis. However, real algal biomass productivity achieved
Per oxygen evolved, four electrons are channeled into so far in the laboratory or small-scale systems does not
the linear photosynthetic electron transport chain and exceed the 73–146 t dry weight haÀ1 yearÀ1 (20–40 g
in the process three molecules of ATP and two of dry weight mÀ2 dayÀ1) and 3% of solar-to-biomass
NADPH are produced. In order to convert three conversion efficiency [25] in the best cases. Currently,
carbon dioxide molecules into glyceraldehyde-3- commercial exploitation of algae mainly utilizes open
phosphate, nine ATP and six NADPH are required. ponds. They are usually raceway cultivators driven by
The ratio is 9.5 mol photons for the conversion of paddle wheels or unstirred, operating at water depths
1 mol CO2 into biomass. The average insolation, that of 15–20 cm, biomass concentration can be up to 1,000
is the solar radiation energy on a surface area in a given mg LÀ1 and productivity between 60 and 100 mg LÀ1
time, is between 3 and 5 kWh mÀ2 dayÀ1 (full solar dayÀ1 (10–25 g mÀ2 dayÀ1) [26]. Commercial rates
spectrum) at temperate regions. However, only about of production of Chlorella have been reported to be
40% of solar radiation is photosynthetically active 60–75 t dry weight haÀ1 yearÀ1 (17–20 g dry weight
(PAR), because only photons with wavelengths between mÀ2 dayÀ1) [27]. Another example is Dunaliella salina,
400 and 700 nm (visible spectrum) can be absorbed by which is now being cultivated on a commercial scale
photosynthetic pigments, since these wavelengths carry and in pilot-scale projects to extract b-carotene and
an energy equal to the change in the energetic level glycerol. Over short periods and in small-scale, pro-
between the ground state and the excited one. ductivity records 60 g dry weight mÀ2 dayÀ1. However,
A photon has an energy that is directly proportional commercial production requires larger culture systems
to its frequency and inversely proportional to its wave- and is presently carried out in outdoor ponds (approx-
length, following Eq. 2. imately 20 cm deep for 5 ha of surface, total ponds area
of 50 ha), because of the still expensive constructing of
E ¼ hn ¼ c =l ð2Þ
closed photobioreactors. In these large-scale systems,
(h, Planck constant, 6.626 Â 10À34 Js; c, light speed, the maximum productivity achievable is at present
3 Â 108 m sÀ1; n, frequency; l, wavelength) 30–40 g dry weight mÀ2 dayÀ1 but more regularly is
For example, 1 mol photons (1 Einstein) at 440 nm below 30 g dry weight mÀ2 dayÀ1 [28]. Outdoor pond
has energy of 272 kJ, while 1 mol photons at 670 nm has cultures of Cyclotella report a biomass productivity of
energy of 178 kJ. Out of the full solar radiation of 12 g mÀ2 dayÀ1 [29]. With the present knowledge and
5 kWh mÀ2 dayÀ1, the photosynthetically active radia- with the available algal strains, real productivity is far
tion (PAR) is about 35 mol photons mÀ2 dayÀ1. Con- below theoretical estimations, especially in large-scale
sidering the energy requirement of 9.5 mol photons per and over long-lasting periods, and a major future goal
1 mol CO2, with such average available light radiation would be to get rid of or reduce this gap.
the cell could assimilate 3.68 mol CO2 mÀ2 dayÀ1. As an alternative to open ponds, few relatively
Since biomass composition can be approximated to large-scale closed photobioreactors have been devel-
the formula CH2O, the previous data would translate oped (refer to “Large-Scale Systems” section for a
in the synthesis of about 110 g biomass mÀ2 dayÀ1. comparison between open ponds and photobioreactors
However, accumulation of organic carbons as biomass in terms of advantages), displaying a productivity of
is lower, due to respiration and other metabolic activ- 2.7 g LÀ1 dayÀ1 in a small undular row tubular
ities and energy losses are accounted to 30%. The photobioreactor of 11 L, 1.9 g LÀ1 dayÀ1 in a airlift
resulting expected maximum biomass productivity tubular photobioreactor of 200 L and 0.05 g LÀ1 dayÀ1


in a parallel tubular photobioreactor of 25,000 L [30]. absorbed light energy in excess with respect to the satu-
Constructing of photobioreactors is still at the ration level of photosynthetic electron transport does
beginning and better productivities (up to ten not contribute to biomass accumulation, but it is
times higher g LÀ1 dayÀ1) as compared to open instead wasted as heat and/or leads to the formation
ponds are obtained with pilot systems. It is likely that of reactive oxygen species (ROS) that ultimately inhibit
such interesting results would also be achieved at larger photosynthesis. Light saturation constant is defined as
scale, by improving photobioreactor design and the intensity of light at which the specific biomass
mechanics. growth rate is half its maximum value [9]. For example,
One major reason for the real lower biomass pro- light saturation constants of microalgae Phaeodactylum
ductivity with respect to the theoretical calculation is tricornutum and Porphyridium cruentum are, respec-
that photosynthesis light reactions have to fit down- tively, 185 mmol photons mÀ2 sÀ1 [31] and 200 mmol
stream biochemical processes while excess absorbed photons mÀ2 sÀ1 [32], much lower than the maximum
energy is dissipated as heat. Photosynthesis displays outdoor sunlight level that occurs at midday in
a light saturation curve [25] (Fig. 1). equatorial regions, that is about 2,000 mmol photons
At low light intensities, light is the limiting factor mÀ2 sÀ1 [9]. In particular, considering a saturating
for the photosynthesis rate, measured as photosyn- light intensity of 400 mmol photons mÀ2 sÀ1, photo-
thetic oxygen evolution. At increasing light intensities, synthesis would saturate at about 7 a.m. and remain
the limiting factor becomes carbon dioxide fixation. saturated until 5 p.m. [25]. An average of 60% up to
When light irradiance overcomes the rate of the down- more than 80% of absorbed irradiance would be wasted
hill biochemical processes, excess absorbed energy is and not converted into chemical energy during the
dissipated and photosynthetic oxygen evolution reaches course of a sunny day [25, 33]. These energy dissipation
a plateau. If light further increases, beyond the capacity events have an important photoprotective role in the
of photoprotective mechanisms, photoinhibition leads natural environment but reduce the potential growth
to a decrease in photosynthesis rate. Therefore, all the rate in biomass culture conditions.

P

Optimal photosynthesis Heat dissipation Photoinhibition

Pmax

P/2

Light Light intensity
saturation
constant

Algae, a New Biomass Resource. Figure 1
Light saturation curve. Photosynthetic oxygen evolution (P) increases with light intensity linearly until saturation. Pmax is
the maximum photosynthetic rate. P/2 is the photosynthetic oxygen evolution rate at half the maximum level. The light
intensity at P/2 is defined as the light saturation constant. Upon saturation of photosynthesis, when downstream
biochemical processes are limiting, excess absorbed energy is dissipated as heat and if light further increases beyond the
capacity of photoprotective mechanisms, photosynthetic rate decreases because of photosystem II photoinhibition


Domestication and some of their characteristics are far from optimal
for growth in mass culture conditions. A domestica-
Crop species currently widely cultivated are “domesti-
tion process needs to be applied to algal species pre-
cated” strains that evolved from wild ancestors [34].
liminary to industrial applications are attempted.
A domesticated crop has been genetically altered and
In particular, selection of strains with the desired
made into a resource for man through cycles of phe-
properties combined with input of new alleles
notypic selection, breeding, and mutagenesis. In this
through mutagenesis and genetic engineering would
way, man can divert evolution of plants to fit
help to generate strains with improved biomass yield,
agronomical, nutritional, and industrial applications.
oil content, and fuel properties. For example, inser-
A fully domesticated crop cannot survive without the
tional mutagenesis can be used to generate random
help of human mankind; meanwhile, farmers would
insertional libraries to be screened for selected
not work with wild species because farming with wild-
phenotypes.
type (ancestral) genotypes would not be economically
Algal biotechnology primarily utilizes unicellular
sustainable. Domestication began in Middle East
species that can be propagated in the laboratory. For
around 10,500 years ago when humans applied selec-
this reason, a great attention is focused on unicellular
tion to cereals and legumes, modifying morphological
microalgae, more suitable for genetic manipulation
and nutritional traits. During history, all crop plants
and prospects of biofuels production [1]. Algae have
were domesticated in a process that took between
several advantages over higher plants in genetic
decades and several centuries depending on the num-
improvement:
ber of genes involved. For example, modern corn was
domesticated from the wild Teosinte to improve pro- 1. A short life cycle enables to genetically manipulate
ductivity and the ability to grow at high density in them faster than crop plants.
cornfields. High plant densities in fields are not dissim- 2. Because of the usual absence of cell differentiation
ilar to the high algal cell densities that are reached up in and haploid nature of most vegetative stages,
a photobioreactor. In the case of tomato, several muta- microalgae allow faster phenotypic selection.
tions in genes involved, for example, in cell division, 3. Microalgae are small and can be analyzed in large
carotenoid biosynthesis, anthocyanin biosynthesis, and numbers in a Petri dish.
ethylene receptor altered fruit color, weight, shape, and 4. It is conceivable that substantial improvements in
ripening of the wild ancestor to finally obtain the now algae cultivation could be achieved in the future.
commercially cultivated tomato [35]. Indirect conven- In contrast, prospects of improvement in higher
tional approaches are now being supported by recom- plants are less favorable, because current cultivated
binant DNA techniques to directly manipulate crops have already been genetically improved with
genotypes and speed up the process. respect to the wild ancestors and cultivation tech-
The natural biodiversity of algae offers a wide spec- niques are already well optimized. Algae have been
trum of phenotypes and specialized adaptation abilities less explored and exploited in the past and current
that can be exploited for commercial applications. Rel- biomass productivity is likely to be improved.
atively few algal strains have been examined to date, 5. Algae are suitable for growth in photobioreactors,
among all the species available in nature. Strains with offering a confined environment for genetically
very interesting features as accumulators of biomass, modified organisms.
biofuels, or other high value products could be still
In the following sections, examples of targets for
unexplored and search for new strains is for
domestication are described.
sure a valuable strategy to pursue. However, similarly
to crops, biofuel production with wild-type algal spe-
Light Use Efficiency
cies collected from the environment, often proposed
as a “green way” to energy supply, is unlikely to be A main process affecting solar-to-biomass conversion
economically sustainable, because wild-type algal efficiency is light harvesting. The photosynthetic appa-
strains evolved to better adapt to their natural habitats ratus comprises two photosystems (photosystem II and


photosystem I) operating in series. Each photosystem is culture. As a consequence of the light gradient formed
composed of an essential core complex, decorated with across the photobioreactor diameter, the real solar
an antenna system of variable size with functions in radiation to biomass conversion efficiency decreases
light harvesting and photoprotection. Chlorophyll far below the calculated value of 8–10% [25], as also
molecules and other accessory pigments bound to pho- mentioned in the “Present Algal Productivity in the
tosystems act cooperatively in the absorption of incom- Laboratory Versus Large Scale” section. The unequal
ing solar radiation. The ability to assemble large arrays light distribution has another negative consequence:
of light-harvesting complexes has been positively algal cells are stirred into the reactor where they can
selected during evolution since it represents a survival move from suboptimal illumination to strong light
strategy and a competitive advantage in the wild, where within few seconds, without having time to adjust the
light could be limiting [36]. Meanwhile, excess light photosynthetic apparatus [47]. Intermittent light is
conditions are mostly avoided by algal cells through highly stressful and contributes to the oxidative stress
changing their location in the water column. Up to 600 and photosystem II photoinhibition.
chlorophyll molecules can be found in association It was first postulated 60 years ago [48] that a
with photosystem II and photosystem I [25]. Light- truncated light-harvesting antenna size would optimize
harvesting complexes are however not a rigid apparatus light utilization efficiency of microalgae in a mass cul-
and both short-term and long-term mechanisms to ture. Such configuration would require a higher light
adjust the light-harvesting capacity to changing light intensity to reach photosynthesis saturation (refer to
conditions are present in photosynthetic organisms. “Present Algal Productivity in the Laboratory Versus
These include heat dissipation of excess absorbed energy Large Scale” section for definition of light saturation
(non-photochemical quenching, NPQ) [37, 38] in the constant) and would minimize wasteful dissipation of
short term and regulation of photosynthetic gene excess absorbed energy and photoinhibition. In partic-
expression during long-term acclimation [39–41]. ular, a better transmittance of light deeper into the
Growth conditions in large-scale mass culture are culture would be allowed, enabling more cells to con-
very different with respect to those found in the natural tribute to useful photosynthesis and culture productiv-
environment. In photobioreactors, cellular concentra- ity. It was estimated that a reduced optical density could
tion is many orders of magnitude higher than in water improve solar radiation to biomass conversion efficiency
bodies, so as to increase production per volume. In up to three to four times [33]. Figure 2 schematically
these conditions, large-size light-harvesting antenna, compares the behavior of the wild type to a truncated
an advantage in the wild, becomes detrimental for antenna strain, with respect to light distribution and
overall biomass productivity because high optical growth inside a hypothetical tubular photobioreactor.
density of antenna pigments results in self-shading While algae with a truncated light-harvesting chlo-
and light attenuation in deep layers of the culture. rophyll antenna size would be useful in controlled mass
Incident light energy is mostly absorbed by cells at the culture conditions, they are not competitive and do not
surface, exceeding photosynthesis maximum rate and survive in the wild, so are not encountered in nature.
resulting in dissipation and loss of excess photons as Although the advantages of strains with altered optical
fluorescence or heat. Up to 80% of the absorbed pho- properties were recognized long ago, only recently, with
tons could thus be wasted [33]. Moreover, these cells molecular genetics coupled to biophysical phenotype
would be more subjected to photoinhibition, that is screening methods, the problem of their construction
the light-induced inactivation of photosystem II can be addressed. Small antenna mutants can now be
due to photooxidative damage [42, 43], leading to obtained through chemical/UV mutagenesis or inser-
losses in photosynthetic productivity [44]. Meanwhile, tion mutagenesis and tested for their light use effi-
a suboptimal illumination would occur in the deepest ciency. Small antenna strains described earlier carried
layers, where energy consumption by respiration would mutations in pigment biosynthesis. Pigments are
reduce the overall yield [45, 46]. Only the intermediate bound to photosystem protein subunits where they
layers are in conditions for optimal photosynthetic act cooperatively in light harvesting, meanwhile pho-
yield and thus determine the productivity of the mass tosystem subunits require a specific set of bound


Wild type Truncated antenna size strain
Su Heat dissipation Su
Photobleaching nl Less heat dissipation
nl ig
ig Photoinhibition ht and photobleaching in the
ht more exposed layer

Photosynthesis
Light
intensity Growth

Photosynthesis

Schematic representation of a tubular photobioreactor transversal section. In wild type (left), most of light is absorbed by
cells in surface layers. Excess light is transformed into heat by physiologic dissipation mechanisms. An algal strain with
truncated antenna (right) is less efficient in absorbing light, thus allowing penetration of the irradiance deeper into the
culture. As a consequence, a higher fraction of cells is photosynthetically active and accumulates biomass and a smaller
fraction of the incident sunlight energy is dissipated into heat due to the lower number of photons intercepted by
each photosystem. However, since antenna protein subunits, beside light harvesting, have important role in
photoprotection preventing the formation of reactive oxygen species or scavenging them, it is essential that the reduction
in the antenna system does not compromise photoprotection capacity of the strain in use

pigments to be properly folded and assembled. Reduc- but no improvements in biomass productivities were
tion of photosystem II antenna size was indeed observed in either laboratory cultures or outdoor
observed as a consequence of mutations affecting the ponds [29]. Every isolated antenna mutant must be
biosynthesis of chlorophyll b [49, 50] or the biosynthe- tested for its growth performance and oxidative stress
sis of xanthophylls [51], specifically coordinated by resistance before any application. Novel genes need to
light-harvesting antenna subunits, in the model unicel- be found, whose deletion or mutation leads to pheno-
lular green alga Chlamydomonas reinhardtii. However, types of selective downregulation of antenna compo-
photosynthetic antenna components are devoted not nents, retaining gene products with photoprotective
only to light harvesting but also to photoprotection, function. Among available methods for the identifica-
meant as the ability to prevent reactive oxygen species tion of these genes, random insertion mutagenesis is
in the light [52]. Moreover, carotenoids of the xantho- of choice (refer to “Future Perspectives and Technolog-
phyll cycle have multiple roles in photoprotection [53– ical Developments” section for further explanation).
55]. Algal mechanisms of oxidative stress resistance are Once a mutant library has been generated by transfor-
particularly important for growth in photobioreactors, mation with a cassette carrying a selectable marker
especially the capacity to respond to fast alterations in gene, strains with the desired characteristics must be
light intensity that cells can face during stirring of the identified from the pool by high throughput methods.
medium. Therefore, efforts at reducing the antenna Truncated antenna size strains can be selected for
must not compromise the photoprotection ability of a lower chlorophyll fluorescence yield and/or pale
the strain, otherwise the advantage obtained in terms of green appearance and/or lower accessory pigments ver-
light distribution would be worthless. For example, sus chlorophyll a content with respect to wild type. The
pigments mutants of the diatom Cyclotella, without fluorescence screening is performed upon application
fucoxanthin, a xanthophyll synthesized in chlorophyll of a saturating light pulse to dark-acclimated cells.
a and chlorophyll c containing algae, displayed Light excites chlorophylls connected to photosystem
a truncated antenna size and a higher light saturation, II which undergo a transition from an “open” state


with low fluorescence to a “closed” state when all pho- Coordinated downregulation of the entire light-
tosystem II primary acceptors are reduced and fluores- harvesting chlorophyll-binding lhc gene family was
cence level is maximal. The maximum fluorescence achieved by RNA interference technology, at last
parameter depends on the number of chlorophylls resulting in increased efficiency of cell cultivation
bound to photosystem II and its value is therefore under elevated light conditions of 1,000 mmol photons
roughly indicative of photosystem II antenna size. mÀ2 sÀ1. Peak density of RNAi cultures was detected
Thus, mutants with reduced chlorophyll antenna are already after 26.5 h, at which parental strain cultures
expected to show a phenotype with a lower yield of had only been able to grow to 54% of their maximal cell
chlorophyll fluorescence with respect to the wild type densities [59]. However, RNA interference-induced
[56]. At room temperature, chlorophyll florescence phenotypes can become unstable after some time.
derives from photosystem II, since photosystem This technology would be useful to identify antenna
I fluorescence yield is negligible in this condition. Pho- components or modulators that can be depleted with-
tosystem I is more efficient in quenching antenna exci- out compromising photoprotection ability. It should
tation because of a faster turnover rate of the be noted, however, that strains for industrial use
photochemistry reactions and its fluorescence is detect- should be stable over time making deletion/insertional
able only at low temperature (77 K). For this reason, mutants preferable.
room temperature fluorescence intensity is indicative Through insertional mutagenesis of C. reinhardtii,
of the sole photosystem II antenna. Meanwhile, since an interesting mutant, called tla1, was isolated for
antenna subunits bind different pigments with respect a reduced yield of chlorophyll fluorescence. It displayed
to photosystem core complexes, namely chlorophyll b a residual chlorophyll content per cell of about 35–40%
and xanthophylls, analysis of pigments by absorption the wild-type level and a functional chlorophyll
spectra and/or HPLC can detect mutations in both antenna size of both photosystem II and photosystem
photosystem I and photosystem II antenna systems. I reduced to 114 and 160 chlorophyll molecules, respec-
Since chlorophyll biosynthesis in the chloroplast is tively [56]. This mutation allowed to identify a novel
strictly coordinated with the expression of both nucleus gene involved in the regulation of antenna proteins
and chloroplast-encoded chlorophyll-binding proteins, accumulation in algae. The tla1 strain required
a “pale green” phenotype could arise from mutations a higher light intensity for the saturation of photosyn-
affecting directly chlorophyll biosynthesis or from muta- thesis, about twofolds higher than wild type, and
tions impairing expression of chlorophyll-binding pro- showed a higher photosynthetic productivity under
teins, their import into the chloroplast or their assembly mass culture conditions, reaching higher cell densities
into photosystems. Mutations affecting these mechanisms (10 Â 106 cells mLÀ1 vs. 6.4 Â 106 cells mLÀ1) [56, 60].
are expected to yield strains with constitutively reduced Simultaneous reduction of photosystem II and photo-
antenna size, irrespective of the compensatory acclimative system I antenna systems is a desired advantage in
mechanisms which are involved in adapting the light- terms of global optical density of the cell. In contrast,
harvesting function to environmental conditions. the sole reduction in photosystem II antenna, if not
Factors controlling antenna proteins expression at counterbalanced by an adjustment in photosystem
the posttranscriptional level are attractive for genetic II/photosystem I ratio, could decrease the photon use
engineering manipulations [57] and a permanently efficiency possibly because a portion of the light energy
active variant of NAB1, a novel cytosolic RNA binding absorbed by photosystem I could not be efficiently
protein, allowed to reduce photosystem II antenna size utilized in the linear photosynthetic electron transport
by 10–17% via translation repression, finally improv- process [49]. The minimum number of chlorophyll
ing light-to-biomass conversion efficiency in high light. molecules, needed for the assembly of the photosystem
In particular, cell culture densities of the antenna core complexes, has been estimated to be 37 for pho-
mutant in the 200 mL bottles increased within 38 h tosystem II and 95 for photosystem I [61]. This would
from 5.97 Â 105 to 1.2 Â 107 cells mLÀ1, whereas the be the minimum chlorophyll antenna size that would
parental strain reached only 5.72Â106 cells mLÀ1 after allow for assembly of the photosystems and that could
the same time period [58]. be theoretically achieved through mutagenesis and


genetic engineering [25]. Reduction in the antenna that is the light-induced inactivation of photosystem II
systems acquired so far is still above this estimated reaction center. More recently, qI was shown to depend
minimum chlorophyll antenna size and further on the synthesis of zeaxanthin, promoted in excess
improvements could be achieved in the future. light. Zeaxanthin binds to light-harvesting antenna
Although truncation of the antenna size of the proteins where it becomes a quencher for chlorophyll
photosystem unit can contribute to improve optical excited states. Its action relaxes when zeaxanthin
properties of the algal suspension, considering the is released and back-transformed into violaxanthin
important role of the antenna systems also in [63]. A third quenching component, relaxing within
photoprotection, an alternative route to pursue could minutes, has been reported and called qT and proposed
be to reduce the number of photosystems per cell and to depend on the phosphorylation of the outer antenna
thus the pigment content per cell. In algae, acclimation proteins that controls their partition between photo-
to high light could rely more on the reduction of the system II and photosystem I during the process of state
photosystem density rather than on major adjustments 1–2 transition [64].
in the antenna system to prevent overexcitation [62]. Photosynthetic light harvesting in higher plants
This natural light acclimation ability could be is mainly regulated through qE and requires PsbS,
exploited in order to generate strains with a photosystem II subunit [65], which is sensitive to
a constitutive reduced optical density through the pH through two lumen-exposed glutamate residues
identification of involved regulatory factors and their whose protonation is required for qE activation [66].
genetic manipulation. Since PsbS does not bind pigments, it cannot be
a quenching site but rather a pH sensitive trigger [67]
transducing a conformational change to antenna pro-
Non-Photochemical Energy Quenching at the
teins binding chlorophylls and xanthophylls. Changes
Molecular Level
in the mutual distance/orientation of these chromo-
Oxygenic photosynthesis involves highly reactive inter- phores reversibly promote energy-dissipation pro-
mediates, such as singlet excited state of chlorophylls cesses such as transient formation of carotenoid
that can decade into triplet excited state, and may lead radical cations, which decay to the ground state
to the formation of reactive oxygen species (ROS). dissipating energy as heat [68, 69]. Although light-
These by-products can damage the photosynthetic dependent energy quenching is a property of all
apparatus and other chloroplast constituents. The photosynthetic eukaryotes, strong differences in the
potential for damage is exacerbated when the amount underlying mechanisms are apparent. Although genes
of absorbed light exceeds the capacity for light energy encoding PsbS are found throughout the green lineage,
utilization in photosynthesis, a condition that can indicating that the protein was present before the
lead to decreases in photosynthetic efficiency. Non- separation between unicellular green algae and
photochemical quenching (NPQ) is the process of multicellular organisms, a PsbS protein is only
heat dissipation of excess absorbed energy, which is accumulated in green macroalgae and land plants.
aimed at regulating light harvesting and preventing Nevertheless, several algal species, such as Chlorella
overexcitation of reaction centers. The major compo- zofingiensis and Scenedesmus communis, exhibit strong
nent of NPQ, called qE, is the fastest established and light-dependent NPQ independent from light intensity
more rapidly reversible quenching deriving from de- during growth [70] or upon acclimation to high light
excitation of the singlet excited state of chlorophyll in as in Chlamydomonas reinhardtii [71], suggesting
the light-harvesting antenna of photosystem II in other gene products might be involved. The moss
response to a change in thylakoid lumen pH. Excess Physcomitrella patens and algal genomes, but not red
light with respect to the capacity of CO2 fixation pro- algae and cyanobacteria, include LHCSR (stress-related
motes lumen acidification since ATP synthesis is lim- members of the Lhc protein superfamily) genes that are
ited by the low ADP + Pi concentration available. In essential for NPQ [72, 73]. Cyanobacteria and possibly
addition to qE, a more slowly relaxing component of red algae, which have phycobilisomes for light
the NPQ process is known as qI, from photoinhibition, harvesting, perform excess energy dissipation by a


mechanism distinct from qE, which depends on the activities are accounted for about 30% energy losses
Orange Carotenoid Protein (OCP) [74]. Understand- with respect to the light energy absorbed leading to an
ing the mechanisms of NPQ and identification of the estimated solar-to-biomass conversion efficiency of
genes involved is valuable as a target for genetic 8–10% in algae [25]. Biomass accumulation ultimately
improvement of algal productivity and domestication, depends on algal photosynthetic architecture and the
since NPQ controls light stress resistance and energy intricate relationships between oxygenic photosynthe-
losses in an algal mass culture. sis, mitochondrial respiration, and catabolism of
endogenous substrates. In order to improve solar-to-
biomass conversion efficiency, it is essential to recog-
Interconversion and Storage of Photosynthetic
nize bottlenecks in the processes interconnecting pho-
Metabolic Products
tosynthesis to other metabolic pathways to make them
Aside from light harvesting, solar-to-biomass conver- targets for engineering. Figure 3 is a schematic repre-
sion efficiency strongly depends on downhill biochem- sentation of the main anabolic and catabolic pathways
ical reactions influencing photosynthate production influencing photosynthate production and biomass
and utilization. Respiration and other metabolic accumulation.

NA
Lipids D(P Starch
ATP )H
AcetyICOA Gly
col
isis
NA
Catabolism of endogenous substrates NA Sugars D(P
½ O2 + 2H+ D(P )H,
H2O ATP )H AT
P
CO2
Mitochondrial CO2 Proteins
respiration Calvin RuBisCO
NAD(P)H cycle
Malate shuttle
ATP
ATP
NADPH
n
tio

H2
ira

O2
H+
sp

NADP+ ADP + Pi
ore

H2ase
FN
lor

2H + R
Ch

PTOX Fdx

PQ
Cyt Chloroplast thylakoid
PSII PQH2 PSI
b6f membrane
ATPsynthase

PC

H2O ½ O2 + 2H+ 2H+

Metabolic overview. Components of the photosynthetic electron transport chain embedded in the chloroplast thylakoid
membrane are depicted. Black lines mark biosynthetic reactions building up the main constituents of the cell, proteins,
starch, and lipids. Dark gray lines mark catabolic reactions that consume endogenous substrates. PSII photosystem II, PSI
photosystem I, Cyt b6f cytochrome b6f complex, PQ plastoquinone, PQH2 plastoquinol, PTOX plastid terminal oxidase,
PC plastocyanin, Fdx ferredoxin, FNR ferredoxin-NADP+ oxidoreductase, H2ase hydrogenase, RuBisCO ribulose-1,5-
bisphosphate carboxylase/oxygenase


The primary reactions of photosynthesis in algae cell. In particular, it was observed that efficient photo-
and plants occur in the chloroplast thylakoid mem- synthesis depends also on mitochondrial respiration in
brane and are catalyzed by the multiprotein-pigment the light [16, 78, 80, 81].
complexes photosystem II (PSII), cytochrome b6f (Cyt Carbon fixation is influenced by alternative elec-
b6f), and photosystem I (PSI). The photosynthetic tron sinks as well as alternative electron sources. For
apparatus itself is a very flexible system, able to modify example, reducing equivalents derived from catabo-
exciton fluxes within its antenna complexes as well as lism of endogenous substrates can fuel the respiration
electron fluxes among its electron transfer components chain in mitochondria but also the photosynthetic
[75]. Photosystem II and photosystem I operate in electron transport chain, thanks to a respiration path-
series within the photosynthetic electron transport way in the chloroplast. Respiration of the chloroplast,
chain. However, alternative electron transport path- also called chlororespiration, was defined as a
ways could be engaged, as well as alternative electron respiratory electron transport chain in interaction
sources or sinks that ultimately would affect availability with the photosynthetic chain [82] and could have its
of reducing power (NADPH) and ATP for carbon origin in the cyanobacterial endosymbiotic ancestor of
dioxide fixation. As a matter of fact, the net products chloroplasts [82, 83]. This pathway was discovered
of photochemistry, NADPH and ATP, that supply about 30 years ago in algae and originally proposed
the Calvin–Benson cycle, are also used by other meta- to account for the changes in the redox state of
bolic pathways such as nitrate assimilation, lipid, plastoquinone, a photosynthetic electron carrier, in
aminoacids, and pigments synthesis. These different darkness [84]. Chlororespiration includes a NAD(P)
sinks may significantly contribute to modify the ATP/ H-dehydrogenase [85, 86] and a plastid terminal oxi-
NADPH ratio and, as a consequence, in vivo carbon dase PTOX [87–91]. The NAD(P)H-dehydrogenase
fixation [76]. The photosynthetic process is divided enzyme of chlororespiration is possibly involved also
into reactions that provide ATP and NADPH (photo- in recycling photosynthesis-generated NAD(P)H,
chemical light reactions) and reactions that consume establishing a cyclic electron transport around photo-
both compounds (carbon assimilation through the system I [92–94]. This alternative electron transport
Calvin–Benson cycle). The rate of these two phases could be engaged to balance the ATP/NADPH stoichi-
may differ in several orders of magnitude, especially ometry, since the net product is only ATP, in response
in high light when carbon assimilation is limiting the to the requirements of the cell and is controlled by the
overall process, and alternative electron sinks may have redox state of the plastoquinone pool. The strong inter-
an important photoprotective role to consume excess play between mitochondria and chloroplast metabo-
generated reductants. Respiration in mitochondria lism is also evident from the characterization of
can serve as well as a sink for excess photosynthesis Chlamydomonas reinhardtii mutants with defects in
generated reducing power. Complex interactions the mitochondrial electron transport chain: an
between photosynthesis in the chloroplast and respira- enhanced glycolysis, to compensate for the absence
tion in the mitochondria occur because both processes of mitochondrial ATP input, is thought to increase
are linked by common key metabolites such as plastoquinone reduction through chlororespiration
ADP/ATP, NAD(P)H, triose-P, and hexose-P [75, and favor cyclic over linear electron transport in the
77, 78]. The chloroplast continuously communicates chloroplast [80].
its metabolic state to the cell, for instance via the Cell fitness depends on the delicate equilibrium
export of carbohydrates, which are synthesized during between biosynthetic and catabolic reactions, both
photosynthesis. The transport of carbohydrates across essential. In order to use a photosynthetic organism
the chloroplast membrane directly allows to exchange as a biofuel or biomass producer, it is essential to
reducing equivalents through “redox valves,” such as control these intricate metabolic dynamics. Carbon
the malate-oxaloacetate shuttle [79]. Different cellular assimilation rate may be a bottleneck, particularly in
compartments are thus strictly interconnected and high light. Dunaliella salina was shown to upregulate
photosynthesis is influenced by either the requirements key enzymes in the Calvin cycle at high salinity, possi-
of the chloroplast or the metabolic status of the whole bly in order to enhance synthesis of glycerol as osmotic


protector [95]. It is conceivable that improvements in due to the fact that most of the photosystem II antenna
carbon assimilation could be achieved in the future is displaced from photosystem II to photosystem
with the help of genetic engineering. Strategies to I during this process and chlorophyll fluorescence at
achieve this aim could be altering expression and/or room temperature arises mostly from photosystem II.
activity of key enzymes or acting on ATP/NADPH The difference in fluorescence between states 1 and 2 can
availability. be measured with a fluorescence video imaging system
and used for screening mutants deficient in state
transition. In the model plant A. thaliana, states 1 and
Screening for State Transition as an Indirect
2 are induced by illuminating cells with light preferen-
Mean to Select Strains with Altered Redox
tially absorbed by photosystem I and photosystem II,
Metabolism
respectively [102]. In C. reinhardtii cells, however,
The redox state of the plastoquinone pool is known to this procedure is not effective because in this alga
control a process called “state1–2 transition.” Since the the absorption spectra of photosystem II and
light-harvesting antennae of photosystem II and pho- photosystem I antennae overlap. Thus, states 1 and 2
tosystem I have distinct absorption spectra, changes in are achieved in the dark by taking advantage of the
the spectral composition of the incident light can lead ability of the chlororespiratory chain to change the
to unequal excitation of the two photosystems and thus redox state of the plastoquinone pool [96, 97]. State
to a decreased photosynthetic yield. Plants and algae transition and cyclic electron transport around photo-
are able to balance the relative excitation of the photosystem I are controlled by the redox state of the plasto-
systems through state transition, that is the reversible quinone pool. This diffusible electron transport element
migration of a fraction of the light-harvesting antenna is at the crossroad between linear electron transport
of photosystem II from photosystem II in state 1 from water to NADP+ and the chlororespiration path-
to photosystem I in state 2, upon phosphorylation way dissipating excess redox power by reducing O2 to
[96–98]. This process was discovered about 40 years H2O. Thus, plastoquinone redox state is altered either
ago in unicellular microalgae [99,100]. The state tran- by an imbalanced excitation of the photosystems or by
sition process has been widely studied in the unicellular changes in the rate of catabolic degradation of storage
green alga C. reinhardtii, where its amplitude is larger molecules and could therefore serve as a key sensor
than in plants and appears to have a major role in of both the incident photon flux and the cellular ener-
balancing the ATP/NADPH stoichiometry by regulat- getic status. Mutations in state transition can affect the
ing the switch between linear and cyclic electron flow, non-photochemical reduction and oxidation of the
in addition to the redistribution of the excitation plastoquinone, catabolism of endogenous substrates,
energy between photosystems following changes in and upstream reactions involved in feeding reducing
light conditions. Indeed, recycling of electrons around equivalents into the stroma compartment of the
photosystem I was observed in state 2 [92–94]. The chloroplast. Screening for state transition mutants is a
interplay between chloroplast and mitochondria useful strategy to isolate strains with an altered redox
metabolism is strong in algae. The mere inhibition of metabolism that could finally affect photosynthate
state transition was shown to be insufficient to modify generation/storage, growth rate, and biofuel produc-
photosynthesis in the presence of active mitochondrial tion. It represents a mean of domestication alone or in
respiration, in contrast it is essential when respiration is combination with other characteristics.
compromised, revealing the physiological significance
of state transition in the energetic contribution [81].
Accumulation of Biomass as Neutral Lipids
Up to 80% of the excitation energy absorbed by the
photosystem II antenna can be redistributed from Assimilated carbons can be stored in high energy value
photosystem II to photosystem I in C. reinhardtii that metabolites, such as starch, proteins, or lipids, as
is therefore well suitable for screening state transition shown in Fig. 2. Some species of algae are particularly
mutants [96, 97, 101]. Large chlorophyll fluorescence attractive because of their ability to produce high
changes occur during a transition from state 1 to state 2, amounts (20–50% of dry cell weight) of neutral lipids


that can serve as a source of biodiesel. Cellular lipid than genus-specific [2]. Interesting species for biodiesel
metabolism is altered toward the accumulation of production include green algae, such as Botryococcus
neutral lipids, mainly triacylglycerols, under stressful braunii, Neochloris oleoabundans, Nannochloris sp.,
conditions, such as nutrient starvation, salinity, Chlorella sp., and Dunaliella primolecta; diatoms,
nonoptimal growth medium pH, low temperature, such as Nitzschia, Phaeodactylum tricornutum, and
high light, but also during aging of the culture [2]. Cylindrotheca sp.; and members of other algal taxa,
These lipids do not have a structural role in membranes such as Nannochloropsis sp. and Schizochytrium sp.
but serve as a storage form of carbon and energy, [9]. Relatively few algal strains have been examined to
confined in lipid bodies in the cytoplasm. Lipid bodies date, among the species available in nature. The possi-
also occur in the inter-thylakoid space of the chloro- bility to isolate new oleaginous strains with the ability
plast in certain green algae such as Dunaliella bardawil to accumulate high levels of oils with the best proper-
[103]. An attractive oleaginous green alga is ties as fuel is likely to offer substantial improvements in
Botryococcus braunii that produces up to 80% of dry lipid yield of industrial cultures while mutagenesis will
weight of very-long-chain (C23–C40) hydrocarbons further enhance the productivity of natural strains.
similar to those found in petroleum, under adverse Fatty acid composition can vary both quantitatively
environmental conditions [8, 104]. Neutral lipids can and qualitatively with the physiological status and cul-
serve additional physiological roles. Fatty acids synthe- ture conditions. Since oil accumulation is enhanced
sis consumes twice the NADPH required for carbohy- under stress, altering growth medium composition
drate or protein synthesis and may thus provide appears as a strategy to improve oil productivity. For
an electron sink under photooxidative stress [2]. More- example, high carbon dioxide concentrations (>5% v/
over, coordination with carotenoid synthesis has v) in Dunaliella salina [107] or nutrient deficiency such
been observed, in particular carotenoids are seques- as nitrogen deprivation in Chlorella vulgaris [108] can
tered into cytosolic lipid bodies where they function lead to a threefold increase in intracellular lipids.
as a sunscreen to reduce light striking the chloroplast Lipid metabolism has been poorly studied in
[2, 105, 106]. algae. Nevertheless, available data suggest that the
Algae triacylglycerols can be exploited to produce basic pathways are analogous to those experimentally
biodiesel, via esterification of fatty acids. Biodiesel has detailed in higher plants. However, differences are
a three to four times higher energy yield than ethanol; distinguishable:
however, biodiesel still represents a small percentage of
1. While in higher plants organic carbon is translocated
total diesel fuel consumption (1.6% in Europe and
from photosynthetically active tissues to sinks
0.21% in the USA, 2005–2007), while ethanol repre-
for lipid synthesis and storage, in microalgae
sents 5% of US gasoline consumption [23]. Currently,
triacylglycerols accumulation takes place within a
biodiesel is mainly produced from higher plants,
single cell together with photosynthesis.
such as palm and soybean [23]. But while extensive
2. While neutral lipids synthesis is mainly associated
cultivation of crops to supply energetic demands is
to seed development in higher plants, it is triggered
unsustainable, competing with food industry for arable
under stress conditions in algae [2].
lands, oil production per hectare from algae, based on
theoretical calculations, would be 100-fold higher than A better knowledge of lipids synthesis pathways
that of soybean and could meet 50% of present US and regulation mechanisms is needed in order to imple-
transportation demand using less than 3% of available ment genetic engineering strategies for oil production.
cropland [9]. Based on experimentally demonstrated Identification of metabolic differences between oleagi-
biomass productivity, oil yield of microalgae could be nous strains and the species that do not accumulate
136 or 58 t haÀ1 yearÀ1 considering 70% or 30% oil in substantial amounts of lipids is a possible research strat-
biomass, respectively [9]. However, insufficient efforts egy to be realized using comparative transcriptomic,
for the establishment of algae-based biodiesel produc- proteomic, and metabolomic profiles of different strains
tion plants have been made until now. The ability of or the same strain under control versus stressful
algae to produce neutral lipids is species-specific rather conditions. Microarray analysis of Chlamydomonas


reinhardtii transcripts under anaerobic incubation has heterotrophic growth, concomitantly with the increase
been performed [109], revealing fermentative pathways of C16:0 and C18:1 fatty acids in triacylglycerols
that produce acetyl CoA, the substrate for fatty acids [118–120]. Monounsaturated fatty acids are indeed
synthesis. Genes encoding enzymes involved in these suitable for good diesel properties [23].
fermentative reactions represent putative candidates Research of new genes involved in lipid metabo-
for increasing triacylglycerols accumulation [2]. lism could be pursued by random mutagenesis.
The limiting step in fatty acids biosynthesis is the A mutant library represents a biological resource of
reaction catalyzed by the first enzyme of the pathway: novel strains that could include improved biodiesel
acetyl CoA carboxylase (ACCase). The properties of producers. An easy and rapid strategy to screen for
this enzyme have been characterized in Cyclotella mutants with altered lipid content is by the use of
cryptica [110–113], but attempts to alter its expression Nile red [121]. Nile red is a hydrophobic molecule
level did not have effects on lipid production [113]. that emits a significant fluorescence signal when
Some transformants showed two- to threefold higher dissolved in lipids at 565–585 nm, in a spectral region
ACCase expression and activity than wild-type cells; where photosynthetic pigments fluorescence is negligi-
however, no detectable increase in lipid levels was ble. Nile red fluorescence detection could be applied
observed. Overexpression of the endogenous enzyme to screen and compare mutant colonies with respect to
may induce negative feedback so that increased activity the wild type in a microtiter plate. Mutants displaying an
of the ACCase enzyme could be compensated for altered Nile red fluorescence yield and thus altered lipid
by other pathways within the cell [113]. Indeed, feed- content with respect to the wild type can then be studied
back inhibition was reported in higher plants [114], by molecular biology techniques to indentify the
where only a heterologous enzyme was successfully mutated gene and tested for oil productivity.
overexpressed really improving oil content [115, 116].
This approach could be proposed again and plants
Planning an Algal Refinery
genes could be used for overexpression in algae.
Although activity and expression of single enzymes Algae convert solar energy into chemical energy and pro-
could be altered, a more powerful strategy might con- duce biomass through photosynthesis. The process
sist in the identification of transcription factors co- requires light, carbon dioxide, water, and other essential
regulating all the genes of the pathway. nutrients and can be considered renewable. Minimal nutri-
Lipids biosynthesis is strictly interconnected tional requirements can be estimated using the approxi-
to other biosynthetic pathways in a unique global mate molecular formula of the microalgal biomass, that is
network. A Chlamydomonas reinhardtii starch-less CO0.48 H1.83 N0.11 P0.01 [9, 122]. However, inorganic
mutant, defective in the ADP-glucose pyrophosphorylase, elements have to be added in excess because they form
was shown to hyper-accumulate triacylglycerol by complexes in solution that are not bioavailable.
a factor of 10 [117]. A strategy to improve oil synthesis Algal biomass can be exploited in different ways,
in order to produce biodiesel could be as well to act on providing several products and biofuels. A scheme
the partitioning of carbon and energy between different of the possible organization of an algal refinery is
pathways. reported in Fig. 4.
Finally, fatty acids composition of triacylglycerols Upon harvesting of the biomass, water and
influences diesel quality and must be considered for nutrients can be recycled, especially using closed
commercial application. Genetic engineering can addi- photobioreactors. Then, biodiesel is obtained from
tionally serve to alter the fatty acids profile thus esterification of fatty acids contained in triacylglycerols
improving fuel properties in engines [23]. Many algae or from other neutral hydrocarbons accumulated by
can synthesize very long polyunsaturated fatty acids in oleaginous algae. A by-product from esterification
large amounts (arachidonic C20:4, eicosapentaenoic of fatty acids to produce biodiesel is glycerol that can
C20:5, docosahexaenoic C22:6 acids), that are extracted serve as fermentative substrate. For example, alga
as high added value products. Polyunsaturated fatty Schizochytrium limacinum produces docosahexaenoic
acids decrease in nutrient-limited medium and during acid, a polyunsaturated o3 fatty acid with beneficial


Glycerol as fermentative
substrate
Water/nutrients
Extraction of
Biodiesel
neutral lipids
H2

Purification of
Light/CO2 Biomass Downstream
Biomass chemicals and
Water/nutrients harvesting processes
pharmaceuticals
CO2

Anaerobic
Electric Biomethane digestion
power
Animal food
(high protein contant)
H2
Fertilizers
(high nitrate contant)

Scheme of the possible utilizations and products of an algal refinery

effects for human health, through fermentation on nitrate. Biomethane (or biogas) has a high energetic
crude glycerol [123]. Currently, algae are already com- yield and is produced by anaerobic bacteria during
mercially used to extract chemicals and pharmaceuti- fermentation of the biomass. The obtained gas contains
cals and this utilization can be combined with the need methane (50–75%) but also carbon dioxide (25–50%).
for biofuels. High added value products derived from However, released carbon dioxide is equivalent to the
algae include polyunsaturated fatty acids, proteins, bio- carbon that algae have assimilated during growth, lead-
polymers, polysaccharides, pigments, vitamins, and ing to zero net carbon dioxide emissions. Carbon diox-
antioxidants [1, 4]. To date, commercialized algae are ide released during the process can be immediately
not transgenic, but this scenario could change in the recycled for alga growth. Electric power produced
near future. For instance, algae can be transformed in from the biogas can sustain the energy demands of
“cell factories” with the help of molecular biology tech- the photobioreactor plant itself [10]. Some algae have
niques for manufacturing recombinant proteins with been evaluated for biomass conversion to methane, in
pharmaceutical or other applications, an approach also particular the giant kelp Macrocystis pyrifera because of
called “molecular farming.” For example, expression of its high growth rate and ease of harvesting [5]. Alter-
human antibodies and vaccines has already proven to natively, algal biomass can serve as fermentative sub-
be successful [124–128]. In general, algae are attractive strate for heterotrophic or photosynthetic anoxygenic
as expression systems for the short life cycle, the cheap sulfur bacteria that use electrons coming from organic
and easy large-scale cultivation, and the ability to grow carbons to produce molecular hydrogen. In particular,
up to high cell densities [129]. Algae have been shown photosynthetic anoxygenic sulfur bacteria use light
free of human pathogens and toxins and thus consid- energy to drive a fermentative reaction and organic
ered safe. acids are electron donors for molecular hydrogen evo-
After extraction of oils and other products, the lution. Generated hydrogen is a high energetic fuel and
spent biomass can serve as animal food or used as clean since its combustion generates water [130].
fermentative substrate for other microorganisms In addition, algae have directly been evaluated for
whose metabolic by-products include biomethane the production of biohydrogen. As a matter of fact,
and biohydrogen. Efﬂuents from the digestion can be some algae are able to evolve hydrogen by a hydroge-
used as fertilizers because of their high content in nase enzyme that catalyzes reduction of protons to


molecular hydrogen. Hydrogen metabolism is primar- hydrogenase and to deplete competitive electron
ily the domain of bacteria and microalgae. Microbial sinks. An improved hydrogen evolution under sulfur
hydrogen formation is catalyzed by either nitrogenases starvation was observed in a mutant affected in the
or hydrogenases, enzymes that can only function under mitochondrial respiratory chain and simultaneously
anaerobic conditions. Nitrogenases are used by certain impaired in the ability to activate state transition
cyanobacteria and photosynthetic bacteria, whereas [16]. In this mutant, accumulation of starch in the
green algae use hydrogenases [12]. In Chlamydomonas chloroplast supplied the photosynthetic electron
reinhardtii, the hydrogenase is expressed in anoxia transport chain through chlororespiration, while
together with the enzymes of the fermentative metab- downregulation of cyclic electron transport around
olism that is active in the dark [109]. However, algae are photosystem I enabled a greater fraction of electrons
attractive for hydrogen production if photosynthetic to be used by the hydrogenase (see “Interconversion
electron transport can be directly exploited, since and Storage of Photosynthetic Metabolic Products”
hydrogen would be generated from the most abundant and “Screening for State Transition as an Indirect
of the natural resources, sunlight and water. Interest- Mean to Select Strains with Altered Metabolism” sec-
ingly, after a period of anaerobic incubation in the tions for further explanations about state transition
dark, photosynthetic hydrogen evolution is detected and cyclic electron transport). The hydrogen produc-
transiently upon illumination [131]. The hydrogenase tion rates of the mutant were 5–13 times higher than in
is transiently active during the dark-to-light transition, wild-type strain, yielding about 540 mL of hydrogen
to consume reducing power in the time needed to fully per 1 L of culture over a 10–14-day period (up to 98%
activate the Calvin–Benson cycle, and receives electrons pure) [16]. However, sulfur deprivation is deleterious
from ferredoxin reduced by photosystem I. This activ- for the cell and this system is sustainable only for few
ity lasts until oxygen evolution by photosystem II days. Upon a recovery phase of sulfur repletion, and
restores aerobiosis and inhibits the hydrogenase reconstitution of reserves as endogenous substrates,
enzyme. In order to photo-produce hydrogen on hydrogen production can be resumed. Nevertheless,
large scale, the major challenge to overcome is the continuous hydrogen production is not possible using
inhibition by oxygen evolved by photosystem II during this technology. An alternative two-stage approach to
oxygenic photosynthesis. Up to now, encouraging overcome the oxygen sensitivity of the hydrogenase,
hydrogen production rates have been obtained by with respect to sulfur deprivation, involves the control
a two-stage approach: the severe oxygen sensitivity of of photosystem II expression [132]. The initial require-
the hydrogenase is circumvented by temporally sepa- ment for this application is a photosystem II-less
rating photosynthetic oxygen evolution and growth mutant which would be complemented by an inducible
(stage 1) from hydrogen evolution (stage 2). A transi- cassette to switch on/off photosystem II activity
tion from stage 1 to stage 2 is performed upon sulfur and oxygen evolution. However, the most efficient
deprivation, which reversibly inactivates photosystem method to obtain large-scale hydrogen production is
II and oxygen evolution. Under this condition, oxida- believed to be the continuous production of hydrogen
tive respiration by the cell in the light depletes oxygen concomitantly to photosynthesis and growth. The
and causes anaerobiosis in the culture, which is neces- limiting factor for such a technology is the finding of
sary and sufficient for the induction of the hydrogenase a hydrogenase enzyme with sufficient resistance to
[13, 15]. Electrons for hydrogen production originate oxygen. Two different approaches are possible: to engi-
from the residual activity of photosystem II as well as neer existing hydrogenases by introducing mutations
from the consumption of endogenous substrates that conferring resistance to oxygen or to find organisms
generates extra electrons supplied to the photosyn- that can synthesize hydrogen even in the presence of
thetic chain through the chlororespiration pathway some oxygen, thus having hydrogenases less oxygen
[16, 86]. Hydrogen production depends from available sensitive. Such hydrogenase could then be heterolo-
electrons sources and competitive sinks of reducing gously expressed in algae [130] to generate
power and a strategy to improve its evolution would a hydrogen producing strain with increased yield of
also be to increase electrons channeling toward the this biofuel.

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