Review

Microalgae and biofuels: A promising

partnership?
F. Xavier Malcata1,2,3
1

ISMAI-Instituto Superior da Maia, Avenida Carlos Oliveira Campos, 4475-690 Avioso S. Pedro, Portugal
CIMAR/CIIMAR-Centro Interdisciplinar de Investigacao Marinha e Ambiental, Rua dos Bragas n8 289, 4050-123 Porto, Portugal
3
Instituto de Tecnologia Qumica e Biologica, Universidade Nova de Lisboa, Avenida da Republica, P-2780-157 Oeiras, Portugal
2

Microalgae have much higher lipid yields than those of

agricultural oleaginosous crops, and they do not compromise arable land. Despite this, current microalga-based
processes suffer from several constraints pertaining to
the biocatalyst and the bioreactor, which hamper technologically and economically feasible scale-up. Here, we
briefly review recent active research and development
efforts worldwide, and discuss the most relevant shortcomings of microalgal biofuels. This review goes one step
further relative to related studies, because it tackles otherwise scarcely mentioned issues for example, heterotrophic versus autotrophic metabolism, alkane versus
glyceride synthesis, conduction versus bubbling of CO2,
and excretion versus accumulation of lipids. Besides
promising solutions that have been hypothesized and
arise from multidisciplinary approaches, we also consider
less conventional ones. Microalgae and biofuels hold
indeed a promising partnership, but a fully competitive
technology is not expected to be available before the end
of this decade, because the need for one order of magnitude increase in productivity requires development of
novel apparatuses and transformed cells.
Microalgal biodiesel versus other fuels
The current appetite of human societies for energy has
driven a critical fossil-fuel dependence; therefore, interest
in microalga-derived biofuels has been on the rise triggered by crude oil price peaking, energy security, greenhouse gas emissions, and competition for otherwise foodoriented agricultural commodities. Microalgae have strong
potential as vectors for biofuels, because their oil content
may exceed 70% (w/wDW), as compared with 5% of the best
agricultural oil crops. Further considerations regarding the
use of microalgae as biofuel sources are put forward in Box 1.
The partnership between microalgae and biofuels is
promising because of the intrinsic autotrophic capacity
of the former. Microalgae require only water and atmospheric CO2, coupled with sunlight, which are available at
essentially zero cost. However, improved strains are urgently needed in terms of volumetric productivity, appropriate lipid profile, and metabolic convenience. Improved
photobioreactors in terms of operation under high cell
densities, gaseous mass transfer, and transmittance of
light, coupled with less expensive downstream processing
are also in order. The issues to be addressed demand a
Corresponding author: Malcata, F.X. (fmalcata@ismai.pt).

542

multidisciplinary approach that encompasses, for example, fundamental biology, systems biology, metabolic
modelling, strain development, bioprocess engineering,
scale-up, biorefineries, integrated production chain, and
whole system design (including logistics) [8]; such an approach is outlined in the following sections.
Selected microalgae with biodiesel production potential
are listed in Table 1. A few differences are apparent among
various species, and even within the same genus. The oil
content is maximum in Botryococcus braunii, but the
associated productivity is poor, and most is secreted into
the cell wall [10]. The most common microalgae (viz.
Chlorella, Dunaliella, Isochrysis, Nannochloris, Nannochloropsis, Neochloris, Nitzschia, Phaeodactylum and Porphyridium spp.) possess oil levels between 20 and 50% (w/
wDW), and exhibit reasonable productivities; Chlorella
appears indeed a particularly good option for biodiesel.
Marine microalgae are more appropriate for large-scale
production because their lipid productivities are consistently higher; furthermore, high salinity prevents extensive contamination and circumvents the need for fresh
water. Other advantages of microalgae over terrestrial
plants are discussed in Box 2.
Unfortunately, the genomes of only three microalgae
have been resolved and annotated to date: Chlamydomonas reinhardtii [13], Thalassiosira pseudonana [14] and
Phaeodactylum tricornutum [15]. Hence, regulation of gene
expression is hardly elucidated, and specific molecular
biology tools, for example, efficient nuclear transformation,
availability of promoter and selectable maker genes, and
stable expression of transgenes are essentially lacking.
Sophisticated metabolic engineering has so far been possible with only C. reinhardtii, despite successful transformation of around 10 distinct microalgal species to date [16].
The desirable features for biodiesel production are
constrained by several bottlenecks toward large-scale,
economically feasible operation; a convenient SWOT
(strengths, weaknesses, opportunities and threats) analysis is briefly depicted in Table 2.
The strengths and opportunities account for the choice
of microalgae as preferential actors in biodiesel production;
and the technological bottlenecks that materialize in weaknesses and threats need a rational approach, encompassing intrinsic and extrinsic parameters playing a role in
microalgal metabolism. The specific microalga strains to be
selected for biodiesel production should thus address the
following issues in a balanced manner [4,17]: (i) optimum

0167-7799/$ see front matter 2011 Published by Elsevier Ltd. doi:10.1016/j.tibtech.2011.05.005 Trends in Biotechnology, November 2011, Vol. 29, No. 11

Review

Trends in Biotechnology November 2011, Vol. 29, No. 11

Box 1. Biofuels instead of fossil fuels

According to the International Energy Agency World Energy Outlook,
the demand for energy will probably increase by 40% between now and
2030. Demand for liquid biofuels tripled between 2000 and 2007;
biofuels contribute to 10% of the global energy supply, whereas liquid
biofuels account for a mere 1.5% of transport fuels. There are four
major types of commercial biofuels: (i) biohydrogen, obtained from
lignocellulosic biomass via microbial photolysis and fermentation; (ii)
biogas, obtained from agricultural and animal waste, or lignocellulosic
biomass via anaerobic digestion; (iii) biobutanol and bioethanol,
obtained from sugar, starch crops and lignocellulosic biomass via
saccharification and fermentation; and (iv) biodiesel, obtained from oil
crops and lignocellulosic materials. The former two still face major
safety issues encompassing storage, transportation and supply;
whereas biobutanol and bioethanol require distillation for concentration that constrains the net energy released besides being hygroscopic, thus easily deteriorating explosion engines. Although the
amounts of biodiesel obtained from oil grain crops and bioethanol
from sugarcane have been increasing [1], their large-scale production is
not sustainable or even feasible. Oil crops can provide some biodiesel
aimed at blending with petroleum diesel to just a few percent, but are

versus enhanced growth, so that structured models and

objective optimization is to be sought instead of empirical
approaches; (ii) alkane versus glyceride synthesis, so that
both the lipid quantity and quality are considered; (iii)
conduction versus bubbling of CO2, to maximize sequestration besides nitrogen and phosphorus uptake; (iv) heterotrophic versus autotrophic metabolism, to take
advantage of nutrients in wastes and effluents; (v) trans-

incapable of providing the paramount quantities of biodiesel necessary

to displace eventually all petroleum-sourced transportation fuels [2]. Oil
palm (probably the most productive oil crop) yields < 6000 l/ha; to fully
supply the USA at present, around 110 million ha would be required
annually that is, an unrealistic 60% of its agricultural cropping.
However, the microalgal productivity can easily attain 1.5 kgbiomass/m3/
day in Mediterranean regions; for an oil content of 30% (w/wDW), the
yield would be 120 m3oil/ha. The 500 million m3 of biodiesel required
by transportation in the USA would thus require microalgae to be
grown on 5.4 million ha that is, just 3% of said cropping area. At
present, the price of crude oil is 100 US$ per barrel; microalgal
biomass with an average 55% (w/wDW) oil content should be produced
at a cost below 340 US$/tonne to be competitive with petroleum diesel,
instead of the current 3000 US$/tonne [3]. Hence, a conservative factor
of almost 10 needs to be overcome so that at least three orders of
magnitude in the scale of production can be attained [4]. Implementation of microalga-based systems has so far been economically
constrained by their poor volumetric efficiency, so biotechnological
improvements are crucial via genetic engineering of microalgae
(which is still in its infancy [5,6]) and via process engineering [7].

mission versus incidence of light, to maximize photosynthetic efficiency and energy savings; (vi) integration versus
separation of processing, to decrease otherwise high costs
of downstream handling; (vii) excretion versus accumulation of lipids, to facilitate recovery of product(s); and (viii)
environmental versus economic sustainability, so that all
relevant inputs are considered in biodiesel pricing. We
briefly consider each of these points in turn.

Table 1. Typical ranges of lipid content and productivity of selected marine and freshwater microalga species [9]
Microalga species
Botryococcus spp.
Chaetoceros calcitrans
Chaetoceros muelleri
Chlorella emersonii
Chlorella protothecoides
Chlorella pyrenoidosa
Chlorella sorokiniana
Chlorella vulgaris
Chlorella spp.
Chlorococcum spp.
Dunaliella primolecta
Dunaliella salina
Dunaliella tertiolecta
Dunaliella spp.
Ellipsoidion spp.
Haematococcus pluvialis
Isochrysis galbana
Isochrysis spp.
Nannochloris spp.
Nannochloropsis oculata
Nannochloropsis spp.
Neochloris oleoabundans
Pavlova salina
Pavlova lutheri
Phaeodactylum tricornutum
Scenedesmus obliquus
Scenedesmus quadricauda
Scenedesmus sp.
Spirulina platensis

Lipid content
(%, w/wDW)
25.075.0
14.639.8
33.6
25.063.0
14.657.8
2.0
19.022.0
5.058.0
10.057.0
19.3
23.1
6.025.0
16.771.0
17.567.0
27.4
25.0
7.040.0
7.133.0
20.056.0
22.729.7
12.053.0
29.065.0
30.9
35.5
18.057.0
11.055.0
1.918.4
19.621.1
4.016.6

Lipid productivity (mg L-1d-1)

Natural habitat

17.6
21.8
10.350.0
1214
44.7
11.240.0
18.7-42.1
53.7
116.0
33.5
47.3
37.8
60.976.5
84.0142.0
60.976.5
90.0134.0
49.4
40.2
44.8
35.1
40.853.9
-

Fresh water
Fresh water
Fresh water

Fresh water
Fresh water

Fresh water
Fresh water
Sea water
Sea water
Sea water
Sea water
Sea water
Sea water
Fresh water

Sea water

543

Review
Box 2. Competitive advantages of microalgae over higher
plants
Conventional terrestrial plants are hardly efficient in capturing solar
energy: switchgrass (the fastest growing one) can convert solar
energy to biomass at no more than 1 W/m2/year, which represents < 0.5% of the total solar energy received in typical midlatitudes [11]; microalgae may attain up to 10%. Their typical
doubling time (24 h) is significantly faster than that of any oil crop,
and essentially independent from weather (provided that a minimum of sunshine hours exists). They require even less water than
land crops for irrigation: 1.5 versus 10 000 l of water to produce 1 l
of biofuel [12]. Plain seawater supplemented with commercial
nitrate and phosphate fertilizers, and a few extra oligoelements
(often available in effluents of aquaculture) are the only nutrients
required, because atmospheric CO2 is available for free (100 tonnes
of microalgal biomass can sequester 183 tonnes of CO2, which
implies a negative ecological footprint), and their halophilic
character brings about ecological advantages against fast-growing
microorganisms (e.g. bacteria).

Optimum versus enhanced growth

Growth rates and productivities have been used for several
decades as metrics for selection of microbial strains and
optimization of environments based on in vitro experiments. Substrates are typically provided in excess and
cells are dilute, so growth rates are theoretically limited
only by the nature of the strain. However, commercial
applications often introduce further complications, such
as limitation of light [18] and nutrients [19]. For common
genera, there is no significant relationship between specific
growth rate and productivity maxima; production of biomass does not necessarily rely on cell division, because
accumulation of lipids also plays a role [20].
A well-established framework to model accurately and
compare fairly the metabolism of different strains under
distinct culture conditions is needed; statistically based
experimental designs are possible [21,22], yet more funda-

Trends in Biotechnology November 2011, Vol. 29, No. 11

mental and integrated strategies are recommended for

example, based on the Dynamic Energy Budget (DEB)
theory, which balances and ascribes value to the various
forms and levels of energy throughout the whole metabolic
cycle. Two types of state-variables, reserve and structure,
are considered, determined by microalgal metabolism, and
consisting of assimilation (i.e. conversion of substrate into
reserves), dissipation (i.e. maintenance and maturation of
structure) and growth (i.e. conversion of reserve into structure); model parameters are directly related to energy and
mass fluxes, and thus have a biological meaning [23]. DEBbased modelling of the microalga Thalassiosira weissfloggi
[24] included photosynthesis and photoinhibition; carbon,
nitrogen and phosphorous uptake; and accumulation and
product synthesis/excretion. Once productivity is defined
(e.g. amount of lipids produced per cell per unit time),
reliable predictions under distinct culturing conditions
are possible, which require specific experimental data, measured under saturating, sub-saturating and limiting concentrations of substrates.
Finally, abiotic and biotic parameter control is important in any microalgal cultivation system, which will benefit from kinetic studies that encompass growth and
metabolite synthesis; however, models that incorporate
measures of carbon fixation according to biological variables and that account for scale-up variations (e.g. raceway
ponds) deserve further enquiry [25].
Alkane versus glyceride synthesis
Classical transport fuels are mixtures chiefly of short chain
alkanes (e.g. octane), whereas biodiesel is an even more
complex mixture of esters with moieties contributed by
fatty acids and short alcohols (e.g. methyl stearate). Manufacture of the latter is a rather cumbersome process that
requires metabolic synthesis of triglycerides first, followed

Table 2. SWOT analysis focused on use of microalgae for biodiesel production

- microalgae use sunlight, a form of energy that is
readily available
- microalgae use CO2, a carbon source that is
readily available from atmosphere and flue gases
- marine microalgae withstand highly salted media,
a competitive advantage to most non-halophilic
microbial predators
- microalgae are metabolically plastic, with physiological
state controlling bioactive compound synthesis
STRENGTHS
WEAKNESSES
- most metabolic pathways in microalgae are not fully
known, thus, biochemical optimization is difficult
- regulatory basis of metabolic networks in microalgae
is incipient, so rational control of endocellular
reactions is hard
- recovery of microalgae from their growth media is
difficult, owing to their small size and planktonic distribution
- several interesting metabolites are endocellular,
but cell wall breaking is difficult and leads to
unspecific mixtures

544

- CO2 is a major component of flue gases, and accounts for global

warming via greenhouse effect
- induction of microalgal secondary metabolism is easy, as several
forms of stress stop growth and trigger lipid synthesis
- coordinated efforts have elucidated the genome of a few microalgae,
thus, development of metabolic engineering tools is likely

OPPORTUNITIES
THREATS
- atmospheric CO2 is excessively dilute and poorly soluble in water,
thus, biomass and lipid yields by microalgae are low
- microalgal uptake of CO2 leads to unwanted pH increases, owing
to conversion to H2CO3 and ionization afterwards
- sunlight is difficult to drive into high-density microalgal cultures,
as required to keep capital costs of occupied land and reactors tractable
- available media for microalgae have not been designed toward
specific metabolite synthesis, so trial-and-error with generic media
is standard practice
- microalgal biomass left after recovery of metabolites should not be
disposed of or incinerated, otherwise it will constitute a vector of
pollution
- focus only on processing steps has left out relevant interactions
with environment, thus compromising validity of life cycle assessments
- economic assessment should encompass the whole biorefinery
approach, as willingness to pay for goods and willingness to accept
environmental impacts are part of the decision process

Review
by chemical hydrolysis and finally reesterification of the
free fatty acids with volatile alcohols.
Metabolic engineering can increase the yields of acylglycerols, or even lead to synthesis of molecules with better
performance as biodiesel. In higher plants, studies have
focused on the effects of overexpression of enzymes associated with lipid synthesis [26], but little change in oil
content has been achieved in species containing high levels
of acetyl-CoA carboxylase the rate limiting step in fatty
acid biosynthesis, possibly because of its complex regulation. In common microalgae, lipids are predominantly
present in cell membranes as phospholipids; following
deprivation of nutrients or exposure to excess light, cells
stop growing and start accumulating triacylglycerols in oil
inclusion bodies, which are a more efficient source in terms
of oil extraction than membranes themselves.
Bioinformatics applied to already sequenced microalgal
genomes have revealed essentially similar routes, but
experimental validation of putative enzyme activities is
still necessary [27]. Proteomic analysis has in particular
provided evidence for a major lipid-droplet protein that
regulates oil body size in C. reinhardti [28], whereas a
starchless mutant deficient in ADP-glucose pyrophosphorylase [29] shows increases in triacylglycerol-containing
lipid droplets; larger oil droplets obviously make extraction
easier and more efficient.
The most consistent reports on metabolic generation of
alkanes pertain to cyanobacteria. Heptadecane (and pentadecane, to a lesser extent) result from decarbonylation of
even-numbered fatty acid aldehydes, via a pathway that
encompasses an acyl carrier protein reductase coupled
with an aldehyde decarbonylase [30]. However, B. braunii
could prove useful in this regard [31] after cloning of the
gene that encodes hydrocarbon biosynthesis. Hence, renewable carbohydrates might be selectively converted to
fuel-grade alkanes, without the need for hydrolysis of
triglycerides followed by reesterification with short-chain
alcohols.
Conduction versus bubbling of CO2
Production of 1 tonne of microalgal biomass requires 1.8
tonnes of CO2 [12], so microalga-based sequestering has
been hypothesized as a practical option to fight greenhouse
effects while bringing about lipid synthesis. However,
release of CO2 by automobiles and trucks occurs in a
scattered fashion, so it would not be feasible logistically
to concentrate and transport it to the bioprocessing plants
[4]; only flue gases from industrial facilities can serve as
feedstock. Increases in biomass and lipid content are regularly observed when CO2 is supplied up to 25% (v/v), but
some strains of Chlorella perform optimally around 10% (v/
v) [32]; wild strains of Scenedesmus obliquus and Chlorella
kessleri have been successfully used for bio-fixation of CO2
in a coal-fired thermoelectric power plant in Brazil [33].
Microalgae can also tolerate up to 300 ppm NOx in flue gas
at sufficiently high initial biomass concentrations [34].
Supply of CO2 in shallow suspensions at near neutral
pH is hardly effective, because the time the bubble remains
in the liquid is insufficient for adequate mass transfer.
Absorption of CO2 is accelerated under alkaline conditions,
with hydration of CO2 and subsequent conversion to

Trends in Biotechnology November 2011, Vol. 29, No. 11

HCO3 (prevailing at pH < 8), and direct reaction with

OH to form HCO3 (dominating at pH > 10) [35]; however,
metabolic performance is suboptimal at extreme pH. The
supply of CO2 diluted in air has classically been accomplished via plain diffusion from the surface of open ponds,
or bulk injection at the bottom of closed reactors, yet only
moderate overall transfer efficiencies (1320%) are observed. Better efficiencies are attained if the gas is conducted through microporous hollow fibres to the inner
portion of the medium; larger specific surface areas, rather
than higher transfer coefficients, make conduction more
efficient in diffusional terms than bulk supply via bubbling.
Thermodynamic constraints arising from the poor solubility of CO2 in water might in addition be alleviated by mixing
an organic solvent (or even an ionic liquid) with the aqueous medium.
The productivity of microalgae is upper bounded by the
rate of photosynthesis and activity of ribulose bisphosphate carboxylase/oxygenase (RuBisCo), the enzyme responsible for carbon fixation. Despite the low affinity of
RuBisCo for its natural substrate [36], microalgae have
adapted to variable (and often-limiting) availability of CO2
and inorganic carbon (Ci) by developing an inducible CO2concentration mechanism. Active Ci uptake leads to extra
internal accumulation, followed by production of elevated
internal CO2 concentrations from Ci via carbonic anhydrase [37]. Genetic engineering to overexpress the active
transporters and enzymes involved therein will probably
enhance environmental CO2 utilization, further to any
increase in solubility and mass transfer.
Heterotrophic versus autotrophic metabolism
Faced with environmental stresses, microalgae store
chemical energy in the form of lipids [27]; carbon accounts
for an average 4555% of cell mass [38], with a typical
overall stoichiometry of C124 N16 P S1.3 K1.7 Mg0.56 Ca0.5
Fe0.0075 Zn0.0008 Cu0.00038 Cd0.00021 Co0.00019. The contents
of N and P, around 7% and 1% (w/w), respectively, appear
mainly in protein, chlorophyll and DNA molecules, and
require twice the amount of fertilizer that is currently
produced in Europe if all transportation fuel were to be
replaced by microalgal biodiesel (so nutrient recycling is a
must).
Phototrophic cultivation (i.e. in the presence of light and
based on photosynthesis) is the most frequently used with
microalgae, and also the easiest to scale up [9]; but several
species can also perform well heterotrophically [39]. Microalgae may assimilate a variety of organic carbon sources,
(e.g. glucose, acetate, glycerol, fructose, sucrose, lactose,
galactose and mannose [40]), thus avoiding light limitation
[41] and leading to higher biomass productivity: Chlorella
protothecoides undergoes a 40%-increase in lipid content
when going from phototrophic to heterotrophic cultivation
[42].
Heterotrophic metabolism offers unique merits viz.,
fast growth and lipid accumulation can be coordinated, and
high lipid yields can be attained [39,40]. Furthermore,
heterotrophic configurations might advantageously use
wastewater and low-cost industrial carbon sources [43]
as nutrients, although carbohydrate-based broths are particularly susceptible to contamination [44], especially in
545

Review
open bioreactors. Finally, development of optimal media
for cultivation is strongly specific for individual species and
intended products, with Fe and Si being usually limiting
micronutrients that have to be added if originally below the
recommended levels.
Transmission versus incidence of light
Availability of light is a sine qua non condition for phototrophic cultivation; it might be just incident light on the
outer surface of the culture container (which penetrates
poorly in the medium, especially under high cell densities),
or be driven into the bulk of the bioreactor after increasing
the transparency of the microalgal suspension or avoiding
surface scattering.
The rate of photosynthesis depends on the photon flux.
At low fluxes, there is a light-limited region in which the
photon input is fully applied in photosynthesis. At higher
fluxes, a light-saturation area is entered, in which the
photosynthetic processing capacity attains its maximum
value, with the excess flux being dissipated as heat or
fluorescence. Photo-inhibition arises afterwards, when further increases in light intensity affect growth rate unfavourably, and eventually lead to culture death. Light
saturation might be overcome by making the supply of
light more uniform, thus driving light closer to the cells; by
tailoring the light spectrum, in terms of quality and temporal characteristics; or by increasing the photosynthetic
efficiency, using genetic engineering [7].
A high surface-to-volume ratio and a shallow depth have
frequently been employed in high cell density photobioreactors to maximize light absorbance and minimize light
attenuation; mixing cycles of a millisecond time scale (i.e.
turbulent eddies) can match the energy captured by microalgal antennae during the short illuminated phase to the
rate of carbon fixation that continues during the dark
phase [43]. Light might also be diluted, by having it strike
closely spaced vertical panels rather than a horizontal one,
on the same land area [45]; or collected externally in a
parabolic solar apparatus and conducted into the reactor
via large-core optical fibre cables, coupled with distributors
inserted between the vertical membrane tubing where
microalgae grow [46]. The oil yield in microalgal ponds
illuminated by sunlight is 100130 m3/ha [2], but it can
reach 172 m3/ha with artificial lighting, primarily because
of its better uniformity. However, the high cost of installing
and operating artificial light sources remains a major
drawback. This might be alleviated by optical fibres exposed to solar energy, if used in combination with solar
panels and wind power generators [47].
Light can be used more efficiently if made available as
short flashes that last for 10 ms (i.e. as close as possible to
the photosynthetic turnover time), separated by longer
dark periods [7]; a few sequential (and crucial) metabolic
reactions do indeed take place in the dark. At high cell
densities, a more intense photosynthetic photon flux helps
photons penetrate deeper into the culture, thus reducing
mutual shading, as well as the ratio of cells receiving
enough light to perform photosynthesis [48,49]. Furthermore, blue and red light is as efficient for photosynthesis as
the full spectrum, so use of light-emitting diodes with those
wavelength widths is recommended [50].
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Trends in Biotechnology November 2011, Vol. 29, No. 11

Table 3. Selected examples of genetic engineering strategies

to reduce microalga antennae [43]
Microalga species
Chlamydomonas
reinhardtii

Chlorella vulgaris
Cyclotella spp.
Dunaliella salina
Scenedesmus
obliquus
Synechocystis spp.

Cell engineering strategy

Random mutagenesis
Targeted mutagenesis
Mutagenesis by DNA insertion
Insertional mutagenesis of Cao gene
Target RNAi repression of all LHC genes
Deregulation of LHC translation repression
Reduced carotenoids
High incident light
Low temperature
High incident light
Mutagenesis by ethylmethylsulphonate
and UV radiation
High incident light
High exposure to CO2
High incident light
Pigment-deficiency

More recent successful strategies have modified strains

aiming at smaller antenna sizes, so that each photosystem
obtains only the light that it needs [51]: shorter antennae
make cells less opaque, thus avoiding wasteful dissipation
of sunlight, while leading to greater photosynthetic productivity [52]. This might be achieved by growing the
microalgae under high light intensity (but the change is
readily reversible), or under nutrient deprivation (but
chlorophyll levels concomitantly fall) [53]. Reduction of
the antenna size can be achieved in a more permanent
manner by mutating the genes responsible for components
of the antenna itself or for its biogenesis [54]; selected
developments in this field are shown in Table 3.
Although small-antenna cell lines might improve the
volumetric productivity [43], cell fitness can meanwhile be
compromised.
Integration versus separation of processing
After lipids have been synthesized and accumulated in
microalgal cells, the biomass should be harvested and
the lipids extracted and purified before eventual transesterification to produce biodiesel. Physical separation steps
might take place downstream, in a sequential fashion, or
take place in situ in the bioreactor, directly on actively
metabolizing cells.
Bulk harvesting, with the purpose of separating the
microalgal biomass from the bulk suspension, is usually
followed by thickening to concentrate the slurry [17]. Harvesting is costly because the cells are small (and thus
possess a low terminal velocity that constrains mechanical
separation); their initial mass fraction is low (so water
removal is expensive because of the major volume reduction), and they normally carry negative charges (that
stabilize them in a dispersed state, and thus hamper
settling) [55]. Thermal drying is expensive, but centrifugation is also energy-demanding [56]. Flocculation, coupled
with sedimentation and flotation, before centrifugation or
filtration might substantially alleviate costs [4], as well as
use of polymer membranes with varying degrees of hydrophobicity to circumvent biofouling.
Harsh conditions are required to disrupt the thick cell
wall of microalgae. The highest lipid yields from Botryo-

Review
coccus spp., Chlorella vulgaris and Scenedesmus spp. are
obtained via bead-beating, microwaving and autoclaving
[57]; however, integrated production of biomass and extraction of metabolites is possible (e.g. n-dodecane enCrypthecodinium
cohnii
growth
and
hanced
docosahexaenoic acid production, but left Scenedesmus
pleiomorphus unchanged [58]).
Ideal lipid extraction should minimize co-extraction of
non-lipid materials, and be selective toward only a few
lipid fractions (e.g. with shorter chain length to promote
volatility [59]). In addition, removal of water is expensive,
so extraction should be applied to the biomass while it is
humid. Hexane is less efficient than chloroform in extracting oils from microalgae, but is also less toxic and has a
residual affinity for non-lipid material [59]. Supercritical
extraction, which usually resorts to CO2 under sufficiently
high pressure and temperature to make it behave somewhat between a liquid and a gas, is a promising technology
owing to tunable solvating power, low solvent toxicity,
and favourable mass transfer rates [60,61]. Its major
drawback is the associated high capital and operating
costs. For instance, lipid extraction yields of 7.1%, 25.0%
and 28.6% (w/wDW) for Chlorococcum spp., Nannochloropsis spp. and B. braunii, respectively [62], were obtained
with supercritical CO2 [75].
Following extraction, triglyceride-rich microalgal oil
has to be converted to biodiesel by transesterification [2]
that is, (reversible) hydrolysis followed by re-esterification with short-chain alcohols (usually methanol) to assure
high volatility, thus generating a mixture of fatty acid alkyl
esters [63]. However, in situ transesterification inside the
biomass itself brought about by a solvent that solubilizes
the reactants and enhances the porosity of the cell membrane, would eliminate the need for biomass harvesting
and extraction: yields found are higher than via the conventional route, and waste is also reduced [64].
Finally, a biorefinery strategy is to be sought throughout the whole process [64]. It should be noted that the
protein resulting from the biomass necessary to supply all
transportation biodiesel in Europe would be 40-fold greater
than the current soy protein imports [4].
Excretion versus accumulation of lipids
Microorganisms can secrete compounds with such physiological functions as nutritional aids or defence tools, yet
lipids accumulate in the cell because they are chiefly
storage components. However, B. braunii secretes alkanes
spontaneously; this concept has been taken advantage of
via genetically modifying microalgae, as pioneered by Synthetic Genomics (La Jolla, CA, USA). Harvesting, drying,
disrupting and extracting microalgal biomass can thus be
avoided because the population actually spends its metabolic energy just to produce and excrete lipids, which are
out-phased with growth. Despite reducing environmental
impacts and improving overall economics [4], this approach does not provide a complete solution because the
residual biomass and other cell components eventually
have to be recovered.
A related possibility is milking microalgae (i.e. harvesting oil directly from diatoms), coupled with a new solar
panel approach [65,66]; this has met with sucess in extrac-

Trends in Biotechnology November 2011, Vol. 29, No. 11

tion of b-carotene from Dunaliella salina [67,68]. In parallel, OriginOil (Los Angeles, CA, USA) patented a specific
electrical modulation of microalgal cells, which increases
their membrane and cell wall permeability. Oil droplets
can accordingly be released without compromising viability, with oil recovery being effected in special two-phase
tanks.
Environmental versus economic sustainability
The industrial viability of microalga-based biofuels hinges
upon their economics; the market will not show any enthusiasm for capital-intensive energy projects unless the
risk/return ratio is acceptable [69]. Major oil companies
worldwide (e.g. Exxon, BP, Chevron and Shell) have taken
up this challenge, and accordingly have made massive
investments in research and development pertaining to
microalgae [70]. It has been claimed that microalgal biofuel will soon become commercially feasible if market
conditions and production technology evolve steadily
[71,72]. For current open ponds, and horizontal tubular
and flat panel photobioreactors, overall processing costs
are 4.95, 4.15 and 5.96 s/kg, respectively. A conservative
conceptual model to assess economics, based on a sensitivity analysis and following historical trends, anticipates
cost reductions to as low as 0.68 s/kg within a decade,
which would then make microalgae a fully competitive
alternative.
The promise of microalga-based biofuels comes also with
the vision of a novel form of large-scale agriculture, probably to be developed in areas that have not previously been
considered for agricultural or industrial uses [73], including desert and offshore marine farming. However, it is
crucial also to consider the associated ecological impacts,
further to regulatory issues, public acceptance and societal
effects. Microalgal cultivation encompasses a large environmental footprint driven by upstream impacts, for example, demand for more concentrated CO2 and fertilizer
[74], and a low energy ratio compared with other biofuels;
hence, pragmatic life cycle assessments (LCAs) are in
order, as described further in Box 3. Such analyses are
likely to be favourable, but require a multitude of parameters to be taken into account.
Although LCA studies applied to aquatic biosystems
have been somewhat controversial [77], microalgal biodiesel proves favourable relative to ultra-low sulphur diesel in
terms of energy and emissions. A negative performance
might result from acidification, eutrophication, toxicity,
land competition and photochemical oxidation [78], as well
as higher energy and water uses, and greenhouse gas
(GHG) emissions, as compared with corn, canola and
switchgrass, for each functional unit of energy produced
[79].
Using flue gas as CO2 source by co-locating microalgal
ponds and coal-fired power plants will not reduce the total
energy consumption and GHG emissions to below those of
terrestrial crops; however, microalgae might be more environmentally beneficial when wastewater is used to supplement (or instead of) chemical fertilizers [79], and good
prospects will be associated with the extra income arising
from several high-added value metabolites of microalgae
[11].
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Trends in Biotechnology November 2011, Vol. 29, No. 11

Box 3. Strategies for environmental impact analysis

The best established tool to perform a product-related assessment is
LCA [75]; it has been used in varying forms over the past four decades
to evaluate the environmental impacts of a product or a service
throughout its life cycle. In essence, this approach analyses the actual
and potential pressure that a product has on the environment during
raw material acquisition, production process, use and disposal of the
product, and provides information to support decisions regarding
product development and ecodesign, production system improvements and product choice at consumer level. Calculation of an
indicator permits sustainability analyses and comparisons between
scenarios for example, Eco-indicator 95, Eco-indicator 99, Ecological
Footprint, Water Footprint and Total Economic Value [76]. For Ecoindicator 95, only effects that damage human health and ecosystems
on a worldwide scale are assessed thus, raw material depletion,
space requirements for waste and local effect are not evaluated. Said
effects (e.g. GHGs, ozone layer depletion, acidification, eutrophication, summer and winter smog, pesticides, heavy metals and
carcinogens) are measured in physical units, namely, mass and
energy, and for any given effect, its weighting is based on the
distance-to-target principle. The Eco-indicator 99 is a state-of-the-art,
damage-oriented impact assessment method for materials and
processes that addresses three damage categories: human health,
ecosystem quality and resources, and minerals and fossil fuels.

Final considerations
To address effectively current bottlenecks associated with
microalgal biodiesel, a leap in both fundamental knowledge and technological applicability is required. After this
has been achieved, microalga-mediated manufacture of
biodiesel will be a fully competitive process [80]. Optimization of lipid productivity should use mechanistic, rather
than empirical models; direct synthesis of alkanes, instead
of glycerides should be promoted; CO2 should be conducted
through porous hollow fibres into the bulk of the medium,
rather than bubbled; heterotrophic metabolism might be
interesting in the presence of large amounts of organic
waste; light absorption by cells should be maximized;
integrated separation of lipids should replace downstream
processing; and environmental considerations should be a
part of any trustworthy economic study.
With regard to biocatalyst engineering, efforts should
focus on: (i) collection of novel genetic resources from microalgae, including genome sequencing to widen the availability of suitable hosts and gene libraries; (ii) improvement of
traditional, and design of novel methods of nuclear transformation; (iii) study of marker and reporter genes, suitable
for controlled overexpression of lipid metabolites; and (iv)
elucidation of metabolic pathways of cellular synthesis and
accumulation/excretion of lipids. For what concerns bioprocess engineering, emphasis should be placed on: (i) design of
novel photobioreactor configurations, specifically targeted
at high rates of mass transfer and light transmission; and (ii)
design and optimization of separation protocols, encompassing harvesting, extraction and purification of lipids.
Acknowledgements
This work received partial financial support from project MICROPHYTE
(PTDC/EBB-EBI/102728/2008), funded by POCI 2010 with the support of
FSE (Portugal), under the coordination of author F. X. Malcata.

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