----------------------- Page 1----------------------Energies 2012, 5, 1532-1553; doi:10.

3390/en5051532
OPEN ACCESS
energies
ISSN 1996-1073
www.m
dpi.com/journal/energies
Article
High Lipid Induction in Microalgae for Biodiesel Production
Kalpesh K. Sharma, Holger Schuhmann and Peer M. Schenk *
Algae Biotechnology Laboratory, School of Agriculture and Food Sciences, The Uni
versity of
Queensland, Brisbane, Queensland 4072, Australia;
E-Mails: kalpesh.sharma@uqconnect.edu.au (K.K.S.); h.schuhmann@uq.edu.au (H.S.)
* Author to whom correspondence should be addressed; E-Mail: p.schenk@uq.edu.a
u;
Tel.: +61-7-3365-8817; Fax: +61-7-3365-1699.
Received: 30 March 2012; in revised form: 3 May 2012 / Accepted: 8 May 2012 /
Published: 18 May 2012
Abstract: Oil-accumulating microalgae have the potential to enable largescale biodiesel
production without competing for arable land or biodiverse natural landsca
pes. High lipid
productivity
of dominant, fast-growing algae is a major prereq
uisite for commercial
production of microalgal oil-derived biodiesel. However, under optimal gro
wth conditions,
large amounts of algal biomass are produced, but with relatively low lipid
contents, while
species with high lipid contents are typically slow growing. Major advance
s in this area
can be made through the induction of lipid biosynthesis, e.g., by environm
ental stresses.
Lipids, in the form of triacylglycerides typically provide a storage funct
ion in the cell that
enables microalgae to endure adverse environmental conditions. Essentially
algal biomass
and
triacylglycerides compete
for photosynthetic assimilate and
a reprogramming
of
physiological pathways is required to stimulate lipid biosynthesis. There
has been a wide
range of studies carried out to identify and develop efficient lipid induc
tion techniques in
microalgae such as nutrients stress (e.g., nitrogen and/or phosphorus star
vation), osmotic
stress, radiation, pH, temperature, heavy metals and other chemicals. In a
ddition, several

genetic strategies for increased triacylglycerides production and inducib

ility are currently
being developed. In this review, we discuss the potential of lipid inducti
on techniques in
microalgae and also their application at commercial scale for the producti
on of biodiesel.
Keywords: algaculture; biofuels; biodiesel; induction; lipids; microalgae
; oil production;
triacylglycerides
----------------------- Page 2----------------------Energies 2012, 5
1533
1. Introduction
Sustainable
production
of renewable
energy
is being
deb
ated
globally
since it is increasingly
understood that first generation biofuels, primarily produced from
food crops and mostly oil seeds,
compete for arable land, freshwater or biodiverse natural landscapes and are lim
ited in their ability to
achieve targets for biofuel production. These concerns have increased the intere
st in developing second
and third generation biofuels such as lignocellulosics and microalgae, respectiv
ely, which potentially
offer great opportunities in the longer term and do not need to compete for arab
le land and precious
freshwater
[1,2]. Due
to continuous
and
increasing
combustio
n
of fossil carbon,
the amount
of
greenhouse gas CO2
has increased. As a result global warmin
g and climate change are threatening
ecological stability, food security and social welfare [3,4]. The transportation
and energy sector are the
two major sources, responsible
for the generation of 20% an
d 60% of greenhouse gases (GHG)
emissions, respectively, and it is expected that with the development of emergin
g economies the global
consumption of energy will rise considerably and this will lead to more environm
ental damage [5].
Photosynthesis is the only process that can convert CO into organic compoun
ds with high energy
2
content, and thus can provide a source for sustainable transpor
t fuel production. There is an urgent
need to develop technologies which are able to produce an additional five to six
billion tons of organic
carbon apart from the current harvest from agricultural crops [3]. Large-scale c
ultivation of microalgae
may be 1020 times more productive on a per hectare basis than other biofuel crops
, are able to use a
wide variety of water sources, and have a strong potential to produce biofuels w
ithout the competition
for food production [2]. Algae can be produced either as macrophytes via marine
aquaculture [6] or in
large-scale microalgae cultivation systems in open ponds or in photobioreactors
[1]. Microalgae are

currently considered the most promising types of algae for biofuel production, b
ased on their high lipid
contents. Recent progress in the production of microalgae has
been intensively reviewed [7], and
future perspectives have been presented by Stephens et al. [5]. Microalgae can a
lso be cultivated as an
integrated concept with wastewater treatment to optimize the ene
rgetic and financial input for the
production process [8].
Triacylglycerides (TAGs) generally serve as energy storage in microalgae tha
t, once extracted, can
be easily converted into biodiesel through transesterification reactions [3,9].
These neutral lipids bear a
common structure of triple esters where usually three long-chain
fatty acids (FAs) are coupled to a
glycerol
molecule.
Transesterification
displaces
glycerol
w
ith small
alcohols
(e.g., methanol).
Recently, the rise in petroleum prices and the need to reduc
e greenhouse gas emission has seen a
renewed interest in large-scale biodiesel production [10].
Within the last few decades the concept of lipid induction
in microalgae has been intensively
studied to increase TAG production in microalgae, but at present different lipid
induction techniques
have not been compared to each other. Here we provide a review of different lipi
d inducing techniques
in microalgae and their potential to be used for biodiesel production.
2. Lipids in Microalgae
Lipids produced by microalgae generally include neutral lipids, polar lipids
, wax esters, sterols and
hydrocarbons, as well as prenyl derivatives such as tocopherols, carotenoids, t
erpenes, quinines and
pyrrole derivatives such as the chlorophylls. Lipids produced by microalgae can
be grouped into two
----------------------- Page 3----------------------Energies 2012, 5
1534
categories, storage lipids (non-polar lipids) and structural lipids (polar lipid
s). Storage lipids are mainly
in the form of TAG made of predominately saturated FAs and some unsaturated FAs
which can be
transesterified to produce biodiesel. Structural lipids typically have a high c
ontent of polyunsaturated
fatty acids (PUFAs), which are also essential nutrients for aquatic animals and
humans. Polar lipids
(phospholipids) and sterols are important structural components of
cell membranes which act as a
selective
permeable
barrier for cells and organelles.
These
lipids maintain
specific membrane
functions, providing the matrix for a wide variety of metabolic processes and pa
rticipate directly in
membrane
fusion events. In addition to a structural function,
some polar lipids may
act as key
intermediates
(or precursors
of intermediates)
in cell signalin
g
pathways
(e.g., inositol lipids,

sphingolipids, oxidative products) and play a role in responding to changes in t

he environment.
Of the non-polar lipids, TAGs are abundant storage products, which can be ea
sily catabolized to
provide
metabolic
energy
[11]. In general, TAGs
are mostly
synthesized
in the light, stored in
cytosolic lipid bodies, and then reutilized for polar lipid synthesis in the dar
k [12]. Microalgal TAGs
are generally characterized by both, saturated and monounsaturated
FAs. However, some oil-rich
species have demonstrated a capacity to accumulate high levels
of long-chain polyunsaturated fatty
acids (PUFA) as TAG [13,14]. A detailed study on both accumulation of TAG in the
green microalga
Parietochloris
incisa and
storage into chloroplastic
lipids (
following
recovery
from
nitrogen
starvation) led to the conclusion that TAGs may play an addit
ional role beyond being an energy
storage
product
in this alga [13,15]. Hence,
PUFA-rich
TAGs
are metabolically
active and are
suggested
to act as a reservoir for specific fatty acids. In re
sponse
to a sudden change
in the
environmental condition, when the de novo synthesis of PUFA may be slower, PUFArich TAG may
donate specific acyl groups to monogalactosyldiacylglycerol (MGDG) and other pol
ar lipids to enable
rapid adaptive membrane reorganization [15,16].
3. Methods of Lipid Induction
The
ability of microalgae
to survive in diverse
and extre
me
conditions
is reflected in the
tremendous
diversity
and sometimes
unusual
pattern
of ce
llular lipids obtained
from
these
microalgae [17]. Moreover, some of these microalgae can also modify lipid metabo
lism efficiently in
response to changes in environmental conditions [12,18]. A review of microalgal
lipid metabolism has
recently been published [19]. Under optimal growth conditions, la
rge amounts of algal biomass are
produced but with relatively low lipid contents (Figure 1), which constitute abo
ut 520% of their dry
cell weight (DCW), including glycerol-based membrane lipids. Essen
tially, microalgae biomass and
TAGs
compete
for photosynthetic
assimilate
and a reprogrammin
g
of physiological
pathways
is
required to stimulate lipid biosynthesis. Under unfavorable environmental or str
ess conditions many
microalgae alter their lipid biosynthetic pathways towards the formation and acc
umulation of neutral
lipids (2050% DCW), mainly in the form of TAG, enabling micro
algae to endure these adverse
conditions (Figure 1).
----------------------- Page 4----------------------Energies 2012, 5
1535
Figure 1. Lipid induction in microalgae under stress co

ndition.
Lipidinducingstre
ssconditions:
Nutrientsstarvat
ion
Temperature(low,hi
gh)shift
Changeinsalinityan
dpH
LightorUVirradiati
on
Microalgaecell

Accumulationoflipi

divisionphase

bodies(yellowdro

d
plets)
asastressrespon
se
High
capital costs due
to low lipid productivity
of
FA-s
ynthesizing
microalgae
are a major
bottleneck, hindering the commercial production of microalgal oilderived biodiesel. One who has
grown microalgae under laboratory or outdoor condition is well aware of the fact
that to obtain high
lipid content,
external stress or lipid induction
techniques
n
eed to be applied. Many
microalgae
produce saturated and unsaturated FAs naturally under ideal growt
h conditions, which have high
nutritional value, but are less ideal for biofuels. However, the synthesis of ne
utral lipids in the form of
TAG can be induced in many species under stress conditions, and these lipids are
suitable precursors
for biodiesel production [20,21]. The occurrence and the extent
to which TAGs are produced is
species/strain-specific, and are ultimately controlled by the genetic make-up of
individual organisms.
Synthesis and accumulation of large amounts of TAGs accompanied
by considerable alterations in
lipid and FA composition can occur in microalgae when placed under stress condit
ions imposed by
chemical or physical environmental stimuli, either acting individually or in com
bination [21]. There
has been a wide range of studies carried out on lipid induction techniques in mi
croalgae such as the use
of nutrients stress, including nitrogen and/or phosphorus starvation, light irra
diation, pH, temperature,
heavy metals and other chemicals. The following paragraphs review
the different TAG induction
techniques and discuss their potential in different microalgae species.
3.1. Nutrient Starvation
Nutrient availability has a significant impact on growth and propagation of
microalgae and broad
effects on their lipid and FA composition. Environmental stress condition when n
utrients are limited,
invariably cause a steadily declining cell division rate. Surprisingly, active b

iosynthesis of fatty acids

is maintained in some algae species under such conditions, prov
ided there is enough light and CO2
available for photosynthesis [12]. When algal growth (as measured by cell divisi
ons) slows down and
there is no requirement for the synthesis of new membrane compounds, the cells i
nstead divert and
deposit fatty acids into TAG. Under these conditions, TAG produc
tion might serve as a protective
----------------------- Page 5----------------------Energies 2012, 5
1536
mechanism. Under normal growth
by photosynthesis are

conditions, ATP

and

NADPH

produced

+
consumed by generating biomass, with ADP and NADP eventually being available ag
ain as acceptor
molecules
in photosynthesis.
When
cell growth
and prolifera
tion
is impaired
due to the lack of
+
nutrients, the pool of the major electron acceptor for photosynthesis, NADP , c
an become depleted.
Since
photosynthesis
is mainly
controlled
by the abundance
of light, and cannot
be shut down
completely, this can lead to a potentially dangerous situation for the cell, dam
aging cell components.
NADPH is consumed in FA biosynthesis, therefore, increased FAs
production (which in turn are
+
stored in TAGs) replenishes the pool of NADP under growth-limiting conditions [
12,21].
Nutrient
starvation
is one of the most
widely
used and
applied
lipid induction
techniques
in
microalgal TAG production and has been reported for many species (Table 1). For
example, when the
diatom Stephanodiscus minutulus was grown under silicon, nitrogen
or phosphorus limitation, an
increase in TAG accumulation and a decrease of polar lipids (as percentage of to
tal lipids) was noticed
in all of the nutrient-limited cultures [22]. In the green al
ga Chlamydomonas moewusii, nutrient
limitation resulted in decreased PUFA C16:3, C16:4, and C18:3
contents whereas overall levels of
C16:1 and C18:1 FA were increased [23].
Nitrogen is the single most critical nutrient affecting lipi
d metabolism in algae. A general trend
towards
accumulation
of lipids, particularly
TAG,
in respon
se
to nitrogen
deficiency
has been
observed in numerous species or strains of various microalgae
[2426]. Hu et al. [27] conducted a
study on nitrogen stress responses of several green microalgae,
diatoms and cyanobacteria and all
tested species showed a significant rise in lipid production. A detailed and lar
ge-scale model of lipid
induction by nutrient starvation (nitrogen, phosphorus) on several
diatoms, green algae, red algae,

prymnesiophytes and eustimatophytes is presented in a study carried out by Rodol

fi et al. [28]. In the
diatom Cyclotella cryptica, higher levels of neutral lipids (primarily TAG) and
higher proportions of
saturated and mono-unsaturated FAs were produced due to silicon deficiency [20].
However, only a
small increase in TAG levels (from 69 to 75% from total lipids) together with ph
ospholipids (from 6 to
8%)
was reported
for the microalga Phaeodactylum
tricornu
tum
as a result of reduced
nitrogen
concentrations
[29]. Scenedesmus
sp. subjected
to nitrogen
or phosphorus
limitation
showed
an
increase in lipids as high as 30% and 53%, respectively [30]. Lipid content of f
reshwater green alga
Chlorella vulgaris could be significantly increased by 40% in low nitrogen-conta
ining medium [31].
With manipulated culture conditions of 1 mM KNO , 1.0% CO , 60 mol photon m2 s1 and
25 C,
3
2
lipid production
of C. vulgaris was
increased
by 2.5 fold
[32]. In addition,
lipid stimulation
in
Chlorella was also achieved via silicon deficiency [33] and iron supplementation
[34]. Moreover, it
was found for C. vulgaris that changing from normal nutrient to nitrogen deplet
ion media gradually
changed
the lipid composition
from free FA rich
lipids to
lipid mostly
contained
as TAG
[35].
Nitrogen starvation in microalgae not only affects the fatty acid metabolism, bu
t also affects pigment
composition. For Parietochloris incise grown in nitrogen replete medium a consid
erable increase in
the ratio of carotenoid and chlorophyll contents was recorded [36].
Phosphorus
limitation
resulted in increased
lipid content,
mainly
as TAG,
in P. tricornutum,
Chaetoceros sp., Isochrysis galbana and Pavlova lutheri, but decreased lipid con
tent in Nannochloris
atomus and Tetraselmis sp. [37]. Due to phosphorus deprivation, production of C
16:0 and C18:1 was
increased and production of C18:43, C20:53 and C22:63
as decreas
ed [37]. In contrast, for
phosphorus-starved cells of the green alga Chlorella kessleri, an
elevated level of unsaturated fatty
----------------------- Page 6----------------------Energies 2012, 5
1537
acids in all identified individual lipids, namely phosphatidylcholine (PC), phos
phatidylglycerol (PG),
digalactosyldiacylglycerol
(DGDG),
monogalactosyldiacylglycerol
(MGDG),
and
sulfoquinovosyl
diacylglycerols (SQDG)
ere found [38]. Phosphorus limitation
as also found to
increase the overall
TAG production from 6.5% up to 39.3%
ith a gradual decrease
in eicosapentaenoic acid (EPA)
concentration. The cellular total lipid content increased, mainly due to TAG acc
umulation in Monodus

subterraneus [15]. In studies carried out on other organisms, including higher p

lants, the authors have
also ackno
ledged replacement of membrane phospholipids by non-phosphorus contai
ning glycolipids
and betain lipids under phosphate limitation [39,40].
A study by Sato et al. [17] on sulphur and phosphorus depletion in green alg
a C. reinhardtii sho
ed
that sulphur depletion leads to decrease in SQDG but on the other hand PG
as in
creased by 2-fold,
representing a compensatory mechanism
here lipids containing sulp
hur are substituted by lipids
containing phosphate. When C. reinhardtii
as gro
n in media
ith limited phosph
orus it sho
ed a
40% decrease in PG and also stimulated increase in the SQDG content. Thus, mecha
nisms that keep
the total sum of SQDG and PG concentrations constant under both phosphorus and s
ulphur-limiting
conditions appear to occur [17]. Other studies have also sho
n that sulfur depri
vation led to increased
total lipid content in the green algae Chlorella sp. and C. reinhardtii [41].
Based on the literature revie
ed, it is clear that amongst all nutrient star
vation approaches, nitrogen
starvation technique is most
idely applied and studied in almost all the microa
lgae species that can be
considered for the production of biofuel (Table 1). Nitrogen is
the most gro
th-limiting factor for
eukaryotic microalgae and
ould be one of the first nutrients to be depleted dur
ing algae cultivation. It
is relatively easy to apply controlled nitrogen stress on microalgae by subtract
ing the nitrogen source
in the gro
th media. Moreover all the microalgae species studied so far (Table 1
), seem to increase
TAG production under nitrogen stress. Hence, nitrogen starvation is the most suc
cessful lipid inducing
technique at present. Ho
ever, high lipid production due to nitrogen stress may
take 25 days and is
complemented
ith slo
gro
th rates and lo
cell counts and thus finally effecti
ng the total biomass
and lipid productivity as detailed by Widjaja et al. [35].
Table 1. Examples of different types of nutrient starvation stress
hich
have been studied
to induce lipids in microalgae.
Microalgae species or
ofile after induction
strain
Chlamydomonas
reinhardtii,
in total lipids

Nutrient stress
Reference

Changes in lipid pr

Increase
Nitrogen limitation
[42]

Scenedesmus
amide ratio)
subspicatus
Nannochloropsis
ncreased by 15.31%
oculata

(lipid:

Nitrogen limitation
[43]

Total lipid i

Chlorella vulgaris
ncreased by 16.41%
Chlorella vulgaris
vity of 78 mg/L d
Chlorella sp.
of 53.96  0.63 mg/L d
Phaeodactylum
sed from 69 to 75%
tricornutum

Nitrogen limitation
[43]
Nitrogen limitation
[24]
Nitrogen limitation
[25]
Nitrogen limitation
[29]

Total lipid i
Lipid producti
Lipid productivity
TAG levels increa

----------------------- Page 7----------------------Energies 2012, 5

1538
Table 1. Cont.
Dunaliella tertiolecta
se in lipid fluorescence
Chlorella vulgaris
ncreased by 40%
Chlorella vulgaris
ease in TAG
Chlorella sp.
n of 49.16  1.36 mg/L d
Chlorella sp.
uctivity of 0.124 g/ L d
Neochloris
ivity of 0.133 g /L d
oleoabundans
Scenedesmus sp.,

Nitrogen limitation
[44]
Nitrogen medium
[31]
Nitrogen limitation
[35]
Nutrient-deprived
conditions (nitrogen,
phosphate-potassium,
[25]
iron, and all three
combined)
Urea limitation
[26]
Ammonium nitrate
[45]

Five times increa

Lipids i
Incr

Total lipid productio

Total lipid prod

Lipid product

Combined effect of
Incre

ase in TAG
Coelastrella sp.
Phaeodactylum
tricornutum ,
ipids
ith higher relative
Chaetoceros sp.,
f 16:0 and 18:1
Isochrysis galbana
Monodus subterraneus
ease in TAG
Scenedesmus sp
and 53%, respectively
Chlorella sp.
Chlorella kessleri
n unsaturated FAs
Chlamydomonas

[46]
Ph and N-limitation
Increase in total l
Phosphorus limitation
[37]
content o
Phosphorus limitation
[15]
Nitrogen and

Incr
Lipids increased 30%

[30]
phosphorus starvation
Silicon deficiency
[33]
Phosphorus limitation
[38]
Sulphur limitation

Increase i
PG
as inc

reased by 2-fold
reinhardtii
Chlamydomonas
ease in TAG
reinhardtii
Cyclotella cryptica
ipids from 27.6% to 54.1%

[17]
Sulphur limition
[41]
Silicon starvation
[47]

Incr
Increased in total l

3.2. Temperature Stress

Temperature has been found to have a major effect on the
fatty acid composition of microalgae
(Table 2) [18,48]. A general trend to
ards increasing FA unsaturation
ith decre
asing temperature and
increasing saturated FA
ith increasing temperature has been obs
erved in many microalgae and
cyanobacteria (Table 2) [4952]. It is generally accepted that many of the lipid p
rofile changes alter
the physical properties of membranes so that normal functions (e.g., ion permeab
ility, photosynthetic
and respiratory processes) can continue unimpaired [53]. The mos
t commonly observed change in
membrane lipids follo
ing a temperature shift is an alteration in FA unsaturatio
n [54]. Due to their
geometry,
FAs
double
bonds
cannot

ith carbon-carbon
be as densely
packed
as saturated
FA, therefore the fluidity of membranes containing unsaturated FA is increased.
As membrane fluidity
is decreased
at lo
er
temperatures,
increased
FA
unsaturati
on
provides
an adaptation
to the
changing environment.
Dunaliella
salina has
been
extensively
analyzed
for lo

temperature
modification
of lipid
composition [12]. A temperature shift from 30 C to 12 C increased the level of uns
aturated lipids
significantly by 20% [12]. In Ochromonas danica, as the incubatio
n temperature rose from 15 to
----------------------- Page 8----------------------Energies 2012, 5
1539
30
C, the cell number
per unit volume
of medium

as
increased
thus increasing
total lipid
content [55]. In Chlorella vulgaris and Botryococcus braunii, incre
ased temperature resulted in a
decrease of the relative content of unsaturated intracellular fatty FAs [56]. In
creases in gro
th rate and
total lipid production
ere obtained in Nannochloropsis salina
ith an increase
in temperature [57].
Whereas, a decrease in culture temperature from 25 to 10 C l
ed to an elevation in the relative
proportion of oleate but a decrease in linoleate and stearidon
ic acid (C18:4n-3) in the green alga
Selenastrum capricornutum [58]. In a culture of I. galbana gro
n at 30 C, total l
ipids accumulated at
a higher rate
ith a slight decrease in the proportion of nonpolar lipids [59].
On the other hand, higher

levels
of omega-3
PUFA
-linolenic
cid
(ALA)
nd
doc
oshexenoic
(DHA)
with 
cid
corresponding decrese in sturted, monounsturted, nd linoleic ftty cids w
ere found in the cells
grown t 15 C [59]. Moreover, in the ditom P. tricornutum the highest yields of
PUFA nd EPA per
unit dry mss were 4.9 nd 2.6%, respectively, when temperture ws shifted from
25 C to 10 C for
12 h, with both being rised by 120% compred with the control [60].
Study on the effects of low tempertures in some higher plnts hve lso bee
n shown to increse the
mount of unsturted FAs [61]. Similr results were lso obtined in Chlorell
ellipsoide where the
content of unsturted FA ws incresed by 2-fold. Moreover,  low temperture-
dpted strin of this
lso showed incresed ALA nd, therefore, more unsturtio
species
n in its PG [62]. In the mrine
microlg Pvlov lutheri, significnt chnges in cidic lipid
nd
ftty cid composition hve been
reported for cultures grown t 15 C compred with 25 C [63]. The culture grown t
15 C resulted in
n incresed reltive mount of EPA nd DHA [63]. Vritions
of FA composition with growth
lso studied by Fork et l. on the thermophil
temperture were
ic cynobcterium Synechococcus
lividus [64]. When the growth temperture ws lowered from 55 C to 38 C, the moun
t of sturted
FA C18:0 decresed while the unsturted FAs C18:1 nd C16:1 incresed [64]. In
generl, there ws
n increse in the more fluid lipids in ll of the lipid clsses when the cells
were grown t the lower
temperture [64].
The
cynobcterium
Spirulin
pltensis
nd
eukryotic
microlge
Chlorell
vulgris
nd
Botryococcus
brunii were
studied
for the effect of mbient
temperture
on the composition
of
intrcellulr FAs nd the relese of free ftty cids (FFA) into the medium [56]
. It ws found tht ll of
the bove species studied, regrdless of their txonomic sttus, responded to th
e temperture regime by
similr chnges in their intrcellulr FA composition: the relt
ive content of more unsturted FAs
decresed nd sturted FAs incresed with the elevtion of temperture [56].
In contrst, no significnt chnge in the lipid content ws observed in Chlo
rell sorokinin grown
t vrious tempertures [65]. There ws no effect of tempertur
cidic
e shift on the content of the
thylkoid lipids, SQDG nd PG, in C. reinhrdtii [17]. It should be noted tht o
nly  limited mount of
informtion is vilble on this subject nd tht ll studies were crried t l
bortory scle where it is
very esy to mintin the desired temperture. Thus mintining, decresing or i
ncresing temperture
is fesible only in closed system photobiorectors which re costly when compre
d to open systems.
At present, we re not wre of ny study tht hs highlighted the effect of tem
perture to induce lipids
on lrge-scle cultivtion; but s lipid profiles clerly chnge t different te
mpertures, properties of
lgl-derived biodiesel would lso chnge for different climtes

nd sesons. Different strins or

species my be used for different sesons (e.g., summer or winter strins) nd e
fforts re underwy to
use flue gses nd other het sources to increse lge growth in colder climte
s.
----------------------- Pge 9----------------------Energies 2012, 5
1540
pertures.

Tble 2. Lipid induction in microlge with different tem

pid profile chnge fter

Microlge species or strin
induction
Chetoceros sp.
lipid incresed by 16.8%
Rhodomons sp.,
d production incresed by
Cryptomons sp.,
5, 12.7, nd 21.7%
Isochrysis sp.
respectively
d production incresed by
Nnnochloropsis ocult
14.92%
Isochrysis glbn
ese in neutrl lipids
rted FA ws incresed by
Chlorell ellipsoide
2-fold
Nnnochloropsis slin
Increse in totl lipids
Dunliell slin
se in unsturted lipids
Ochromons dnic
rese in totl lipids
Selenstrum cpricornutum
crese in olete ftty cid
Isochrysis glbn
Increse in totl lipids
Pheodctylum tricornutum
t yields of PUFA nd EPA
Pvlov lutheri
ed reltive mount of EPA

Li
Reference

[49]

Stressing gent

Grown t 25 C

Totl
Lipi

Rnge of 27 C to 30 C
[49]

15.

Lipi
Increse from 20 C to 25 C
[43]
Increse from 15 C to 30 C
[59]

Incr
Unstu

Lowering temperture
[62]
Increse in temperture
[57]
Shift from 30 C to 12 C
[12]
Increse from 15 C to 30 C
[55]
Temperture from 25 C to

Incre
Inc
In

[58]
10 C
Grown t 30 C

[59]
Shifted from 25 C to 10 C
Highes
[60]
[63]

for 12 h
Grown t 15 C

Incres

Spirulin pltensis, Chlorell

vulgris, Botryococcus
Sturted FAs incresed
brunii

Increse in temperture
[56]

3.3. Slinity-Induced Lipid Production

Dunliell
species
provide
the best exmples
of micro
lge
tht cn
tolerte
high slt
concentrtions. The bility of Dunliell species to proliferte over prcticl
ly the sturtion rnge of
slinities mkes them one of the fvorite cndidtes to study slinity effects o
n microlge [6668]. In
 study crried out by Azchi et l. [66] cells of D. slin
were trnsferred from 0.5 to 3.5 M
(29 to 205 g/L) NCl, nd there ws  significntly higher rtio of C18 (mostly
unsturted) to C16
(mostly sturted) FAs in the cells grown in 3.5 M (205 g/
L) NCl compred with those grown t
0.5 M (29 g/L) NCl [66]. An increse of the initil NCl concentrtion from 0.5
M (29 g/L) to 1.0 M
(58 g/L) followed by further ddition of NCl to 2.0 M (117
g/L) during cultivtion of Dunliell
tertiolect resulted in n increse in intrcellulr lipid content nd  higher
percentge of TAG [67].
An even stronger increse in slinity from 0.4 M to 4 M (23 to 234 g/L) in Dunl
iell sp. incresed the
proportion of totl sturted ftty nd monounsturted ftty cids, wheres the
proportion of PUFA
ws decresed [68].
The ditom Nitzschi levis is known to produce high mounts of EPA [69]. Wh
en these cells were
subjected to high slt concentrtions, the degree of FA unstu
rtion of both neutrl
nd polr lipid
frctions incresed shrply when slt concentrtions incresed from 10 to 20 g/L
, but decresed t slt
concentrtions of 30 g/L [69]. Highest contents of totl ftty
cids, EPA
nd polr lipids were ll
obtined t NCl concentrtion of 20 g/L, under which 71.3%
of totl EPA existed in polr lipid
----------------------- Pge 10----------------------Energies 2012, 5
1541

mount
lso
frctions
[69]. The
of totl free sterols ws
n increse
incresed
with
in slt
concentrtion. In three mrine heterotrophic microlge strins, Crythecodinium
cohnii ATCC 30556,
C. cohnii ATCC 50051 nd C. cohnii RJH grown t different slinities, the FA co
mposition ws lso
ffected [70]. At 9 g/L NCl, C. cohnii ATCC 30556 hd the
highest totl FA content
nd DHA
(C22:6) proportion. In contrst, C. cohnii ATCC 50051 nd
C. cohnii RJH hd the highest DHA
content t 5 g/L NCl. C. cohnii ATCC 30556 nd ATCC 50051 hd the highest DHA y
ield (132 nd
68 mg/L respectively) t 9 g/L NCl while C. cohnii RJH hd the highest DHA yiel
d (129 mg/L) t

5 g/L NCl [70]. Growth, lipid content nd FA composition of heterotrophic micro
lg Schizochytrium
t different
limcinum
OUC88
tempertures
(16 C, 23 C,
nd
30 C nd
37 C)
slinities
(0, 9, 18, 27 nd 36 g/L) were
nlyzed [71]. Highest lipi
d content ws obtined t slinities of
936 g/L t  temperture rnge of 1630 C nd the content of sturted ftty cids C
15:0 nd C17:0
ws incresed gretly [71]. In ddition, the rtio of DHA to DPA chnged t diff
erent tempertures nd
slinities [71].
3.4. The Effect of pH nd Hevy Metls Stress

lso hve been foun

Fluctutions of the pH in the medium
d to lter the lipid composition of
microlge (Tble 3). For exmple, lkline pH stress led to TAG ccumultion in
Chlorell CHLOR1
nd ws not dependent on nitrogen or crbon limittion levels,
nd led to 
decrese in membrne
lipids [72]. Bsed on morphologicl observtions, lkline pH inhibited the grow
th of microlge, thus
diverting the energy to form TAG [72]. The effects of pH o
nd FA composition of 
n the lipid
Chlmydomons sp. isolted from
 volcnic cidic lke,
nd C.
reinhrdtii hve been studied nd
compred [73]. In the unidentified Chlmydomons sp., FAs of polr lipids were m
ore sturted thn
those in C. reinhrdtii. The reltive proportion of TAG (s percentge of totl
lipids) ws higher in
t pH 1 thn tht in the cells culti
Chlmydomons sp. grown
t higher pH. The increse in
vted
sturtion of ftty
cids in membrne lipids of Chlmydomons hs
been suggested to represent
n
dptive rection t low pH to decrese membrne lipid fluidity [73].
Hevy metls like cdmium, iron, copper nd zinc hve lso been found to inc
rese the lipid content
in some microlge [74]. The effect of high levels of cdmium ws studied in Eug
len grcilis grown
s utotrophic, heterotrophic (in the drk) nd mixotrophic (in the light with 
n orgnic crbon source)
cultures [74]. Cdmium cused n increse in the totl lipid
ll three culture
content per cell in
systems
[74]. Among
the membrne
lipids, sterol content
ws
lower
in cdmium-treted
cells
cultivted under illumintion. There were no chnges in the totl phospholipid c
ontent, lthough there
ws n increse in PG. E. grcilis hs lso been shown to disply somewht diffe
rent sensitivities to
copper nd zinc [74]. The effect of iron on growth nd lipid ccumultion in Ch
lorell vulgris ws
investigted by Liu et l. [34]. The culture in the lte exponentil growth phs
e when supplemented
with Fe3+ t different concentrtions, showed incresed totl lipid content of u
p to 56.6% biomss by
dry weight [34].
----------------------- Pge 11-----------------------

Energies 2012, 5
1542
Tble 3. Exmples of lipid induction in microlge due to slinity
nd pH stress.
Microlge sp
hnge fter induction
Dunliell slin
ntrtion of C18 FA
Dunliell tertiolect
pid content nd TAG
in totl FA nd
Dunliell sp.

turted FA
Nitzschi levis
n unsturted FA
Crythecodinium.

l FA content nd DHA

cohnii ATCC 30556
Schizochytrium
5:0 nd C17:0 ws
limcinum
tly incresed

Slinity chnge
Reference
Trnsferred from 29 to
[66]
205 g/L NCl
Trnsferred from 29 g/L to
[67]
58 g/L NCl
Incresed slinity from
[68]
23 to 234 g/L NCl
NCl concentrtion
[69]
incresed from 10 to 20 g/L
At 9 g/L NCl
[70]
Slinity t 936 g/L t

tl lipids to 56.6% of

Chlorell vulgris

Incresed conce

Increse in li
Increse

monouns
Increse i

Increse in tot

Sturted FA C1

temperture rnge of
[71]

gre

1630 C

Unidentified
in sturted FAs
Chlmydomons sp.
Chlorell sp.
rese in TAG
Eugleni grcilis
se in totl lipids

Lipid profile c

Low pH
[73]

lkline pH
[72]
Cdmium, copper, zinc
[74]
3+

Increse
Inc
Incre
Increse in to

Fe
[34]

biomss
3.5. Light Irrdition Stress
Light is the most importnt element for photosynthesis,
out which no utotrophic life cn
sustin or flourish. Microlge hve been reported to grow
vrious light intensities exhibiting
remrkble
chnges
in their gross chemicl
composition,

with
on

pigment
content
nd
photosynthetic
ctivity [75] (Tble 4). Moreover, different light intensities n
d wvelengths hve been reported to
chnge the lipid metbolism in microlge ltering the lipid profile [76] (Tble
4). High light intensity
leds to oxidtive dmge of PUFA [76], nd is lso required for the synthesis o
f C16:1 (3 trns) nd
lters the level of this ftty cid in microlge. Typiclly, low light intensit
y induces the formtion of
polr lipids, prticulrly the membrne polr lipids ssocited with the chlorop
lst, wheres high light
 simultneous
intensity decreses totl polr lipid content with
increse in the mount of neutrl
storge lipids, minly TAGs [7780]. High light exposure decresed the totl phosp
holipid content nd
incresed the level of nonpolr lipid (nmely TAG) in the filmentous green lg
Cldophor sp. [79].
In the red microlg Tichocrpus crinitus exposure to low light intensity result
ed in incresed levels of
some cell membrne lipids, especilly SQDG, PG nd PC, wheres higher light inte
nsities incresed
the level of TAG [78]. In the hptophyte Pvlov lutheri higher light intensiti
es ct s  ctlyst to
increse the lipid content nd were ssocited with lower dilution rte-promoted
increses in both cell
popultion
nd weight per cell [81]. TAG production under high
s

light conditions might serve
protective mechnism for the cell. As outlined bove, electron cceptors needed
by the photosynthetic
mchinery might be depleted under high light conditions s well. Incresed FA sy
nthesis which in turn
re stored s TAG, potentilly helps the cell to re-generte its electron ccept
or pool.
----------------------- Pge 12----------------------Energies 2012, 5
1543

ffects the ftty

cid composition
Light intensity not only
in microlge, but
lso the pigment
composition. In the green microlg Prietochloris incise under l
ow irrdince photosyntheticlly
ctive
nd  r
rdition,
cultures
displyed
slow
growth
eltively
low
crotenoid-to-chlorophyll
rtio [36]. At higher irrdinces on complete medium, the lg displyed  highe
r growth rte nd n
increse in the crotenoid content, especilly tht of -carotene and lutein [36].
Light/dark
cycles
at different growth
phases
also hav
e
a significant
effect on
algal lipid
composition, as was successfully demonstrated in a detailed study on various lig
ht regimes on lipids of
the
diatom
Thalassiosira
pseudonana
[77].
A culture
gr
own
to stationary
phase
under
strong
continuous light or under 12:12 h strong light/dark conditions
had a higher amount of TAG with
saturated
and monounsaturated
fatty acids
compared
to c
ultures
grown
with
less light. At the
exponential
growth
phase,
however,
the proportion
of

PUFA
was
highest
under
high
light
conditions [77]. This demonstrates the important role of growth phase in the acc
umulation of certain
fatty acids. With the onset of stationary phase, algae typically show increased
proportions of saturated
and monounsaturated fatty acids and decreased amounts of PUFA [
77]. The lipid and fatty acid
compositions
of three species
of sea ice diatoms
grown
in chemostats
have
een analyzed
and
compared
when
cultivated
at light-limiting
conditions
of
2 and 15 mol
photons
m2 s1 [82].
Growing cultures at 2 mol photons m2 s1 resulted in 50% more MGDG containing EPA th
an those
grown at 15 mol photons m2
s1. Growing cultures at 2 mol
photons m2
s1 resulted in higher
amounts
of non polar
lipid bilayer forming
MGDG
in r
elation
to total bilayer forming
lipids,
especially DGDG (the ratio of MGDG:DGDG increased from 3.4 to
5.7) than in cultures grown at
2 1
15 mol photons m
s . A shorter light period seemed to
increase the production of PUFA in
Isochrysis galbana [83]. Sitosterol and stigmasterol were the two main sterols
detected at 246.3 and
220.0 mg/100 g, respectively. A continuous increase in the level of total sterol
s was recorded during
the life cycle at 24 h lighting [83]. The reduction of the
photoperiod led to a decrease in the total
sterols produced in the decay phase. A gradual increase in -to
copherol production during the life
cycle ws lso recorded [83]. Drk tretment cused  decrese in the reltive p
roportion of olete ftty
cid nd n increse in linolete ftty cid in the green lg Selenstrum cpri
cornutum [58]. In the
dinoflgellte
Prorocentrum
minimum
drk
exposure
led
to  reduced
content
of TAG
nd
glctosylglycerides, while the totl content of phospholipids chnged little [5
8].
Light
irrdition cn only
be controlled
in  closed
sy
stem
biorectors
or in lbortory-scle
cultures, s shown by the exmples bove. Moreover, opertionl costs for contro
lled light dd up to
the production cost of biofuels from microlge, lthough there re severl comm
ercil pproches of
using LEDs or diverted sunlight in lrge-scle photobiorectors. Light is essent
il for TAG production,
but if high light irrdition is used s  stimulnt for incresed TAG productio
n, bsed on the exmples
bove nd in Tble 4, it cn be expected tht this will differ for different spe
cies. In ddition, TAG FA
composition is different for different species in response to different light ex
posures. For exmple, in
Nnnochloropsis sp. the degree of unsturtion of FAs ws lower with
incresing irrdince with 
significnt decrese in omeg-3 ftty cids (29% to 8% of totl FA), cused min
ly by  decrese of
EPA (20:5n-3) [84]. In conclusion, light will normlly stimulte ftty cid synt
hesis, growth nd the
formtion of (prticulrly chloroplst) membrnes. Therefore, the ov

erll lipid content of

lge
reflect such morphologicl chnges.

will

----------------------- Pge 13----------------------Energies 2012, 5

1544
3.6. UV Irrdince for Lipid Induction
Current reserch on the effect of UV irrdince in microlge is minly focu
sed on the impct of
UV-A
nd UV-B rdition on
lgl growth, morphology, physiology
nd oxidtive stress [8589],
with  specil emphsis on pigments nd photosynthesis [90]. Exmples of studies
on UV rdition on
lipid profiles in microlge re shown in Tble 4.
In  study crried out by Srinivs nd Ochs [91] on Nnnochloropsis ocult
the effect of UV-A t
different
levels
of exposure
on totl lipid ccumultion
ws
investigted.
UV-A
tretments
significntly incresed the PUFA (chlorophyll-specific lipid concent
nd
rtion) of N. ocult cells,
nd decresed nutrients hd 
UV-A
synergistic effect on lipid
ccumultion. The effects of UV-B
rdition
on the totl lipid, FA
nd sterol composition
n
d content
of three Antrctic
mrine

phytoplnkton species
Odontell
weissflogii, Chetoceros simplex
nd the hptophyte Pheocystis
were exmined in 
preliminry culture experiment [92
ntrctic
]. The cultures were exposed to
nd low or high UV-B rdition. The sterol, f
constnt UV-A
tty cid
nd totl lipid content for
Odontell weissflogii chnged little under low UV-B when compred
with control conditions. In
contrst, when P. ntrctic ws exposed to low UV-B irrdince,
storge lipids were reduced
nd
structurl lipids incresed [92]. P.
ntrctic
lso contined

higher proportion of polyunsturted
ftty cids under low UV-B exposure. Exposure of P. ntrctic to high UV-B irr
dince incresed
totl lipid, TAG nd FFA concentrtions. Lipid concentrtions per cell lso incr
esed when C. simplex
ws
exposed
to high UV-B
irrdince
[92]. This resulted
from increses
in FFA
concentrtion
principlly
sturted
FA
indicte
degrdtion
of
nd my
complex
lipids during
high UV-B
tretment
[92].
Effect
of UV-B
rdition
on lipid prod
uctivity
ws
studied
in detil in
Tetrselmis sp. [93]. A 4 hour-exposure to UV-B rdition resulted in n overll
increse in sturted
nd monounsturted FA, wheres the PUFA content ws decre
FA
sed by 50% [93]. In ddition,
UV irrdince cused 
decline in the overll rte of crbon
mino
cids
nd

incorported into
mino
cids [93]
reduction in the pool size of totl cellulr
. In contrst, intrcellulr dissolved free
mino cid incresed [93].
The effect of UV rdition on growth nd ftty cid composition of two dito
ms, P. tricornutum

nd Chetoceros muelleri, were exmined in btch cultures [94].

UV rdition induced signi cnt
differences in ll the mjor ftty cids of P. tricornutum. The percentges of E
PA nd PUFA incresed
while monounsturted FA decresed in the UV-A tretment in comprison with no U
V irrdince or
combined UV-A + UV-B tretments [94]. On the other hnd, ll the mjor ftty ci
ds of C. muelleri
vried with hrvest stge nd UV irrdince. The percentge of monounsturted F
A in C. muelleri
incresed, while EPA nd PUFA decresed under combined UV-A + UV-B tretment [94
]. The study
indicted tht UV-A exposure my promote EPA nd PUFA formtio
n in P. tricornutum, wheres
n
combined
UV-A + UV-B
exposure
enhnced
short FA
d monounsturted
FA
content,
but
suppressed PUFA formtion in C. muelleri [94].
PUFAs, especilly EPA nd DHA, re bundntly synthesized by some phytoplnk
ton species nd
ply  key role in the mrine food chin. However, they re generlly considered
to be sensitive to
oxidtion by UV rdition. P. lutheri nd Odontell urit were exposed to  co
mbintion of UV-A
nd UV-B rdition with  totl dily dose of 110 kJ/m2 nd lipid composition w
s then determined on
dys 3, 5, nd 8 of UV exposure [95]. In P. lutheri exposure to UV led to  decr
ese in the proportion
----------------------- Pge 14----------------------Energies 2012, 5
1545
of PUFAs, especilly those in structurl lipids (glycolipids nd phospholipids)
nd  reduction of 20%
in EPA levels nd 16% in DHA levels, fter 8 dys; wheres for O. urit, expos
ure to UV did not
chnge the ftty cid composition of the totl lipids nd lipid frctions of the
cells [95].
Interestingly,
UV
rdition
hs been suggested
for
micro
lipid induction
in lrge-scle
lgl
cultivtion systems. As UV rdition hs geneticlly nd physiologiclly deleter
ious effects on mny
life forms including microlge [95], the impct is conceivbly
relted to the rdition intensity. A
recent study showed tht the modulted use of UV-A rdition
n
for seven dys could led to
incresed production of ftty cids in Nnnocloropsis sp. [96]. However, there i
s concern tht constnt
use of UV-A light my not be vible for industril-scle cultivtion, while sho
rter, but stronger UV
rdition could lso ffect microlgl lipid composition nd production s  res
ult of inhibiting nutrient
uptke, crbon ssimiltion mechnism nd dmging DNA [97].
Tble 4. Lipid induction due to different light irrdition stre
ss in microlge.
Lipid

profile chnge fter

Microlge sp
induction
Tichocrpus crinitus
Incresed TAG
Pvlov lutheri
esed totl lipid content
Thlssiosir pseudonn
Incresed PUFA
Thlssiosir pseudonn
Incresed TAG
Unidentified ditoms
more MGDG

Irrdition type
Reference
Low light intensity
[78]
High light intensities
[81]
Continuous or light/drk
cycled strong light t
[77]
exponentil growth
Continuous or light/drk
cycled strong light t
[77]
sttionry phse
Low light (2 mol photons
2 1
[82]
m
s )

Incr

50%

Selenastrum
crease in linoleate FA
capricornutum
Prorocentrum minimum
increase in phospholipids
Isochrysis galbana
Increase of PUFA
Nannochloropsis oculata
of PUFA, structural lipids
P. antarctica
in PUFA, structural lipids
C. simplex
ncrease in total lipids

Dark treatment
[58]
Dark treatment
[58]
Shorter light period
[83]
UV A
[91]
Low UV B
[70]
High UV B
[70]

In
Marginal

Increase
Increase
I
Inc

rease in saturated and

Tetraselmis sp.

UV B radiation
[93]
m

onounsaturated FA
Phaeodactylum
UV radiation
reased EPA and PUFA
tricornutum
Chaetoceros muelleri
sed monounsaturated FA

Inc

[98]
UV A

Increa

[98]
Increase

in saturated FA to PUFA
Nannochloropsis sp.

UV A
[96]

ratio
4. Genetic Engineering of Microalgae to Increase Lipid Production
Apart from inducing lipid biosynthesis in microalgae by external cues, some
progress is emerging
towards
metabolic
engineering
towards
higher TAG
or ome
ga 3
accumulation
capacities.
The

model
species C.
molecular,
genetic

reinhardtii has been

and physiological

the focus

of most

Page 15
Energies 2012, 5
1546
research [99106]. Significant advances in microalgae genomics have been achieved
during the last
decade [103,104,106]. Expressed sequence tag (EST) databases have been establish
ed; transcriptomes
as well as nuclear, mitochondrial and chloroplastidial genomes fr
om several microalgae have been
sequenced; and several more are in progress of being sequenced
[102]. Of particular relevance in
relation to fatty acid biosynthesis, is acetyl CoA carboxylase (ACC) which was f
irst isolated from the
microalga Cyclotella cryptica in 1990 by Roessler [107] and was later successfu
lly transformed into
the diatoms C. cryptica and Navicula saprophila [108]. ACC1 was over expressed l
eading to 2 3 fold
enhanced enzyme activity. These experiments demonstrated that ACC could be trans
formed efficiently
into microalgae although no significant increase of lipid accumulation was obser
ved in the transgenic
diatoms [108]. It also suggests that over expression of ACC enzyme alone might n
ot be sufficient to
significantly enhance the lipid biosynthesis pathway. For a recent review on the
potential of metabolic
engineering
and
other
genetic
targets
to enhance
lipid
accumulation
in microalgae,
see
Schuhmann et al. [19]. However, it should be pointed out that at present, GM str
ains of microalgae can
only be used in small scale closed bioreactors and are very strictly regulated.
This may increase the
total cost of production when compared to non GM algae in open pond systems.
5. Conclusions and Future Directions
We have discussed the different lipid induction techniques th
at can be used to stimulate lipid
biosynthesis, in microalgae, in particular TAG. It is clear th
at different microalgae species react to
different stresses by producing different fatty acids or by altering their compo
sition of fatty acids. Thus
which
techniques
to apply for the lipid induction
in particu
lar microalgae
might
depend
on the
environmental conditions and cultivation systems. Perhaps the most
obvious way to advance our
understanding of how the environment can alter lipid metabolism in microalgae is
to study one species
under controlled laboratory conditions. Based on the
literatur
e reviewed, it is clear that amongst
different lipid induction techniques, nitrogen starvation is most widely applied
and studied in almost
all the microalgae species that can be considered for the commercial production
of biodiesel (Table 1).
Change in temperature, pH, salinity and heavy metals can also induce lipids effe
ctively, but may be

difficult
to regulate
on a large scale
cultivation
system
(Ta
bles
24). Rapid
induction
of lipid
biosynthesis maybe achieved through hybrid systems where a nutrient rich flow cu
lture in exponential
phase continuously produces algal biomass of which batches (e.g.
, 50%) can be transferred to low
nutrient conditions but with sufficient light irradiance for photosynthesis. The
lipid accumulation phase
maybe further shortened by applying a combination of different induction types.
For example, this may
include a sudden induction, as possible in a hybrid system,
combined with nutrient depletion, a
salinity/pH change as well as exposure to UV irradiance. The exact combination o
f induction stresses
that provides optimum lipid productivity in a large scale commercial cultivation
system for biodiesel
production, will differ for every microalgae strain and depends on nutrient supp
ly, environmental and
climatic conditions.
Page 16
Energies 2012, 5
1547
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osten, C.; Kruse, O.;
Hankamer, B. Second generation biofuels: High efficiency microalgae for b
iodiesel production.
BioEnergy Res. 2008, 1, 2043.
2.
Chisti, Y. Biodiesel from microalgae. Biotechnol. Adv. 2007, 25, 294306.
3.
Chisti, Y. Biodiesel from microalgae beats bioethanol. Trends Biotech. 2
008, 26, 126131.
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Christenson, L.; Sims, R. Production and harvesting of m
icroalgae for wastewater treatment,
biofuels, and bioproducts. Biotechnol. Adv. 2011, 29, 686702.
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