Appl Microbiol Biotechnol (2005) 66: 486–496
DOI 10.1007/s00253-004-1779-z
MINI-REVIEW
P. Metzger . C. Largeau
Botryococcus braunii: a rich source for hydrocarbons and related
ether lipids
Received: 6 July 2004 / Revised: 23 September 2004 / Accepted: 24 September 2004 / Published online: 4 December 2004
# Springer-Verlag 2004
Abstract This paper presents a review on Botryococcus
braunii, a cosmopolitan green colonial microalga charac-
terised by a considerable production of lipids, notably
hydrocarbons. Strains like wild populations of this alga
differ in the type of hydrocarbons they synthesise and
accumulate: (1) n-alkadienes and trienes, (2) triterpenoid
botryococcenes and methylated squalenes, or (3) a
tetraterpenoid, lycopadiene. In addition to hydrocarbons
and some classic lipids, these algae produce numerous
series of characteristic ether lipids closely related to
hydrocarbons. This review covers the algal biodiversity,
the chemical structures and biosynthesis of hydrocarbons
and ether lipids and the biotechnological studies related to
hydrocarbon production.
Introduction
Botryococcus braunii is a green colonial microalga
widespread in freshwater and brackish lakes, reservoirs,
ponds, or even ephemeral lakes situated in continental,
temperate, alpine, or tropical zones (Wake and Hillen
1980, 1981; Aaronson et al. 1983; Huszar and Reynolds
1997; Huang et al. 1999; Metzger and Largeau 1999;
Volova et al. 2003). This alga is characterised by a
conspicuous ability to synthesise and accumulate a variety
of lipids. These lipid substances include numerous
hydrocarbons, i.e. highly reduced compounds comprising
only carbon and hydrogen as elements (Brown and
Knights 1969; Knights et al. 1970), and a number of
specific ether lipids (Metzger et al. 1991; Metzger and
Largeau 1999).
P. Metzger (*) . C. Largeau
Laboratoire de Chimie Bioorganique et Organique Physique,
Ecole Nationale Supérieure de Chimie de Paris,
11 Rue Pierre et Marie Curie,
75231 cedex 05 Paris, France
e-mail: pierre-metzger@enscp.jussieu.fr
Tel.: +33-144-276717
Fax: +33-143-257975
To avoid any confusion, it is important to specify what
kinds of compounds are considered as “lipids” in the
present Mini-Review. According to the classic definition,
lipids are all the compounds produced by living organisms
which are sparingly soluble in water but readily soluble in
organic solvents (Ratledge and Wilkinson 1988); and their
structures may contain straight long hydrocarbon chains or
isoprene units and various functional groups, especially
oxygenated ones. In this paper, we choose to separate B.
braunii lipids by a functional group approach, so that
straight-chain and isoprenoid hydrocarbons are presented
together, while a second section is devoted to ether lipids.
Strains of B. braunii isolated and grown in laboratories
and wild populations of this alga both differ in the type of
hydrocarbons they synthesise. Accordingly, they are sub-
classified into three chemical races. Algae in race A
produce essentially n-alkadiene and triene hydrocarbons,
odd-carbon-numbered from C23 to C33 (Metzger et al.
1985a), algae in race B produce triterpenoid hydrocarbons,
C30–C37 botryococcenes (Metzger et al. 1985a) and C31–
C34 methylated squalenes (Huang and Poulter 1989a;
Achitouv et al. 2004) and algae in race L produce a single
tetraterpenoid hydrocarbon, lycopadiene (Metzger and
Casadevall 1987; Metzger et al. 1990). The chemical
structures of some characteristic hydrocarbons typical of
the three chemical races of B. braunii are shown in Fig. 1.
In addition to hydrocarbons, B. braunii also synthesises
classic lipids such as fatty acids, triacyl glycerols and
sterols; and a list of these and their distributions can be
found in a previous review (Metzger and Largeau 1999).
A second feature of this alga is the production of
numerous ether lipids of a new type which are not
glycerol derivatives, like those occurring in all other living
organisms. In each race, ether lipids are closely related to
hydrocarbons; and in some strains their production can be
largely dominant. Lastly, non-polysaccharide biopolymers
of very high molecular weight (104 Da to 4×106 Da),
polyaldehydes and polyacetals, have been isolated from
lipid extracts of B. braunii. Their occurrence and possible
functions as precursors of the insoluble polymeric material

Fig. 1 Types of hydrocarbons
produced by the three chemical
races of B. braunii (I: Knights et
al. 1970; II: Villarreal-Rosales et
al. 1992; III: Metzger and Ca-
sadevall 1983; IV: Huang and
Poulter 1989b; V: Metzger and
Casadevall 1987)
building up the outer walls of the alga were recently
reviewed by Metzger and Largeau (2002).
Owing to its oil richness and its ability to form blooms,
sometimes enduring over many years, like in the Darwin
Reservoir in Australia (Wake and Hillen 1980; Townsend
2001), this microalga has been proposed as a renewable
source of liquid fuel (Casadevall et al. 1985). Furthermore,
oil production via CO2 fixation could also mitigate the
emission of greenhouse gases (Pedroni et al. 2001).
Several studies were therefore carried out to determine the
optimal conditions for B. braunii culture and hydrocarbon
production.
The present article focusses first on the algal biodiver-
sity, the chemical structures of hydrocarbons and ether
lipids and their biosynthesis and then on some biotechno-
logical developments related to the production of algal
hydrocarbons.
Biodiversity of B. braunii
Mophology and taxonomy
Colonies of B. braunii under microscope observation
exhibit a typical morphology (Fig. 2a) characterised by a
botryoid organisation of individual pyriform-shaped cells
held together by a refringent matrix containing lipids. Oil
droplets can be excreted from the matrix by the pressure of
a coverglass. Ultrastructural studies reveal that the matrix
surrounding the basal part of the cells consists of outer
walls originating from successive cellular divisions (Fig.
2b). Furthermore, the bulk of B. braunii hydrocarbons are
487
stored in these outer walls (Largeau et al. 1980). However,
there exists an important morphological heterogeneity
within algae examined after water-sampling from lakes
and cultivation of strains in the laboratory. The most
striking variations concern the size and shape of cells,
which can be more or less embedded in the matrix, and the
presence (or not) of refringent threads linking clusters of
cells, thus leading to the formation of very large colonies
(Fig. 2c). On the basis of such morphological differences,
but ignoring chemical analyses, Komárek and Marvan
(1992) proposed the existence of at least 13 species in
Botryococcus. However, Plain et al. (1993) noted that, in
each chemical race and for the same strain, some of these
features could vary in relation to age and culture
conditions. Recently, 18S rRNA sequences of four strains
of B. braunii belonging to the three chemical races
established that these strains formed a monophyletic group
(Senousy 2003; Senousy et al. 2004). Now, whether the
numerous strains of B. braunii belong to a single species,
to three species in connection with the nature of the
synthesised hydrocarbons, or to several sub-species is still
under debate.
Hydrocarbon content
Today, a total of about 60 samples of B. braunii, including
strains cultivated in laboratory and wild samples collected
from lakes, have been analysed for their hydrocarbon
content and composition (for a non-exhaustive list, see
Metzger and Largeau 1999). They originate from all
climatic zones, with the exception of the Antarctic. While
488

algae of races A and B have been identified in alpine,

continental, temperate and tropical lakes, those of race L
are only observed in water samples collected in the tropics.
Algae of race A exhibit the largest range in hydrocarbon
content, from 0.4% (Bolivian strain from lake Overjuyo;
Metzger and Largeau 1999) to 61.0% of their dry weight
(French strain from lake Chaumeçon; Metzger et al
1985a). By comparison, in algae of race B the hydrocar-
bon content is generally intermediate, accounting for 30–
40% dry weight (Metzger et al. 1985a; Okada et al. 1995),
but lower levels down to 9% have also been observed
(Okada et al. 1995). The exceptionally high level of 86%
of botryococcenes noticed by Brown and Knights (1969)
with algae in the resting phase of growth, probably due to
partial lysis or degradation of cell content, has not been
observed again. Algae of race L show a hydrocarbon
content ranging from less than 0.1% in two Indian strains
up to 8.0% in a Thai strain (Metzger and Casadevall 1987;
Metzger et al. 1997).
A comparative overview of the relative abundance of
hydrocarbons and some other lipids is given in Table 1 for
three strains of race A and one strain each of races B and
L. Depending on the strain, the content of hydrocarbons
and ether lipids varies significantly.

Hydrocarbons

Chemical variability

Hydrocarbons from B. braunii race A

About 30 chemical structures have been determined

Fig. 2 Micrographs of B. braunii. a Light microscopy of an English
strain (Maddingley Brick Pits, UK), race A: some refringent
globules of lipids are ejected from the colonies by the pressure on
the cover glass. b Transmission electron microscopy of a colony of a
strain from Ivory Coast (Yamoussoukro), race L: the section shows
successive outer walls surrounding the cells. c Scanning electron
microscopy of a very large colony of a Martinique strain (La
Manzo), race B. Scale bars: a, c 10 μm; b 1 μm
among the 50 hydrocarbons detected in cultivated strains
and wild samples of B. braunii race A (Metzger and
Largeau 1999). All these compounds, ranging from
monoenes to tetraenes and almost all odd-carbon-num-
bered, exhibit a terminal unsaturation. The hydrocarbon
distributions are under the control of genetic factors:
variations were observed from strain to strain cultivated
under identical conditions (Metzger et al. 1986). Gen-
erally, dienes predominate; and they comprise a mid-chain
unsaturation, predominantly in a cis configuration (Metz-
ger and Largeau 1999). The most frequently found trienes
exhibit two conjugated mid-chain unsaturations, while the
other, rarely occurring, exhibit two conjugated double
bonds in a terminal position (Metzger 1993). Only one

Table 1 Relative abundances of Compound Race A Race B Race L

some types of lipids in five
strains of the three chemical Bolivia UK Morocco Martinique (La Ivory Coast
races of B. braunii grown in the (Overjuyo) (Maddingley) (Oukaimden) Manzo) (Yamoussoukro)
laboratory (expressed as % dry
wt). For analytical details, see Total lipids 62 63 43 53 35
Metzger (1994, 1999), Metzger
and Casadevall (1992), Metzger Hydrocarbons 0.4 9 20 32 3
and Largeau (1999) and Metz- Ether lipids 35 5 n.d. 0.2 13
ger et al. (1985a, 1990, 2002, Epoxides n. d 4 n.d. 1 0.6
2003). n.d., Not determined Triacylglycerols 2 6 n.d. n.d. n.d.
Sterols n. d 0.1 n.d. 0.2 0.2
 489

type of tetraenes has been identified with three conjugated

double bonds in a terminal position (Metzger 1993).
Incubations with radio-labelled tracers have shown that
oleic acid is the direct precursor of dienes and trienes
(Templier et al. 1984, 1987) and that decarboxylation of
very long chain fatty acid derivatives, activated by a β-
substituent, is the final step leading to the formation of the
terminal unsaturation (Chan Yong et al. 1986). In contrast,
a cobalt-porphyrin decarbonylase isolated from B. braunii
race A (incubated with a fatty aldehyde) yielded an alkane
(Dennis and Kolattukudy 1991, 1992). However, it can be
noted that saturated paraffinic hydrocarbons are not
biosynthesised by living algae.

Hydrocarbons of B. braunii race L

Trs-trs-lycopadiene (Fig. 1) was the sole hydrocarbon

detected in strains from Thailand and Ivory Coast
(Metzger and Largeau 1999), but a second minor isomer
of unknown structure was detected in two Indian strains
(Metzger et al. 1997). Nothing is known on lycopadiene
biosynthesis.
Fig. 3 Various C34 isomeric botryococcenes from race B (I: Cox et
al. 1973; II: Galbraith et al. 1983; III, V: Metzger et al. 1985b; IV:
Hydrocarbons from B. braunii race B Okada et al. 1997a; VI: David et al. 1988)

Botryococcenes, specific hydrocarbons of B. braunii race

B, are triterpenoids comprising acyclic and cyclic
compounds. Out of the 50 botryococcenes identified,
only about 15 structures have been determined, due to
difficulties encountered in their purification (Metzger and
Largeau 1999). The first described was a C34 compound
(structure I in Fig. 3) isolated from a wild sample by Cox
et al. (1973); and today five other C34 isomers are known
(structures II–VI in Fig. 3). The C30 botryococcene (Fig. 1)
isolated from a cultured strain (Metzger and Casadevall
1983) is the precursor of all higher homologous
compounds; and botryococcene distribution is strain-
dependant (Metzger et al 1985a). In addition, algae of
race B synthesise squalene and C31–C34 methylated
squalenes (Huang and Poulter 1989b; Metzger and
Largeau 1999). Generally present in trace amounts, they
were isolated in an appreciable yield from a Bolivian strain
(4.5% of dry biomass; Achitouv et al. 2004).
Biosynthesis of triterpenoid hydrocarbons in race B
The non-mevalonate pathway
Due to the massive production of triterpenes, particularly
botryococcenes, B. braunii race B must efficiently produce
isopentenyl diphosphate (IPP) and dimethylallyl diphos-
phate (DMAPP), the fundamental precursors for isopre-
noid biosynthesis. Until recently, IPP was believed to be
produced in all living organisms from the condensation of
acetyl CoA through the mevalonate (MVA) pathway. In
the case of B. braunii, a labelling experiment showed that
the incorporation level of [2-14C] MVA into botryococ-
cenes was however low (0.2%; Casadevall et al. 1984). In
fact, the 13C-labelling pattern observed after feeding the
algae with [1-13C] glucose established that botryococcenes
and methylated squalenes resulted from the non-MVA
pathway (Sato et al. 2003). In this pathway (Fig. 4),
pyruvate and glyceraldehyde-3-phosphate resulting from
glycolysis are condensed into 1-deoxy-D-xylulose-5-phos-
phate (DOXP), in turn transposed into 2-C-methyl-D-
erythritol-4-phosphate (MEP), the intermediate in IPP
biosynthesis via the non-mevalonate pathway (Schwender
et al. 1996; Duvold et al. 1997).
The presqualene diphosphate pathway
Squalene and C30 botryococcene are non-head-to-tail
triterpenes resulting respectively from 1′-1 and 1′-3
linkages of two farnesyl moieties (Poulter 1990). As
reported by Inoue et al. (1994a, 1995), [1-14C] farnesol
was actually incorporated in a high yield into squalene
(1.3%) and botryococcenes (19.0%) during a feeding
experiment and could be phosphorylated to its mono- and
diphosphate esters with a cell-free extract of B. braunii
race B. However, in vivo incubations of [1-3H] farnesyl
diphosphate (FPP) with a cell-free extract did not confirm
the putative role of FPP as precursor of botryococcenes:
the radio-labelled precursor was incorporated into squa-
lene but not into botryococcenes. This suggested that
another derivative of farnesol was intermediate in
botryococcene biosynthesis (Inoue et al. 1993). However,
490
Fig. 4 Schematic biosynthetic
pathways for botryococcenes
and methylated squalenes. FPP
Farnesyl diphosphate, S-AM S-
adenosyl methionine. Bold ar-
rows indicate the positions of
methylations leading to C31–C34
higher homologues
in a recent study Okada et al. (2004), using a modified
experimental procedure, established that FPP is indeed a
precursor of botryococcenes: Triton X-100 used by Inoue
et al. (1994a) in their experiments is in fact an inhibitor of
botryococcene synthase, while it stimulates the synthesis
of squalene.
Both stereochemical studies (White et al. 1986, 1992)
and incorporation experiments performed with (R)- and
(S)-[1-2H] farnesol (Huang and Poulter 1989b) gave
support for presqualene diphosphate (PSPP) as a common
precursor in the synthesis of squalene and C30 botryo-
coccene. Cleavage of the rearranged cyclopropane leads to
squalene, while direct cleavage of cyclopropane leads to
C30 botryococcene. Today, it is unknown whether two
separate enzymes are responsible for the synthesis of
squalene and botryococcene, or whether a single enzyme
machinery is capable of producing two different irregular
triterpenes. Indeed, it was recently shown that a recom-
binant yeast squalene synthase is able to synthesise a
hydroxybotryococcene from PSPP, in addition to two
squalene derivatives (Jarstfer et al. 2002).
Alkylation and cyclisation
Incubations of the algae with radio-labelled tracers
followed by chase experiments in cold media indicated
that C30 botryococcene is the precursor of its higher
homologues (Wolf et al. 1985a; Metzger et al. 1987).
Experiments performed with L-[Me-14C]- and L-[Me-13C]-
methionine demonstrated that this amino acid, likely via
its S-adenosyl form, is the methylating agent (Metzger et
al. 1987). Given the variability of the chemical structures
of botryococcenes in relation to the strain origin, as
illustrated in Fig. 3, the pattern of the successive
methylations occurring at positions indicated by bold
arrows in Fig. 4 likely depends on genetic factors. In B.
braunii race B, alkylation of squalene proceeds in a similar
way, with methionine as the source of methyl groups for
the synthesis of C31–C34 higher homologues (Huang and
Poulter 1989a; Achitouv et al. 2004). The biosynthetic
pathway of cyclobotryococcenes is presently unknown: it
could be initiated either by protonation of a methylated
botryococcene or by methylation (Metzger et al. 1985b;
Huang and Poulter 1988).
Ether lipids and their biosynthesis
Ether lipids occur widely in Nature, generally as 1-O-alkyl
and 1-O-(1′-alkenyl) analogues of triacylglycerols and
glycerophosphatides, or as isopranoid dialkyldiglycerol
tetraethers in Archaea (Mangold and Paltauf 1983). By
contrast, ether lipids from B. braunii are not glycerol
derivatives, but closely related to the hydrocarbons.
Ethers from B. braunii race A
Initial work revealed the huge production of ether lipids by
two strains of B. braunii race A originating from Bolivia
(lake Overjuyo) and France (lake Coat ar Herno) with the
predominance of an alkadienyl-O-alkatrienyl ether ex-
hibiting an oxygen bridge between two C27 hydrocarbon
chains (compound I, Fig. 5; Metzger and Casadevall
1991). The ether lipid content maximised at 42% (dry
weight) in the exponential phase of growth and then
decreased slowly down to ca. 20% in the stationary phase.
By comparison, hydrocarbons were only minor constitu-
ents in these strains throughout growth (Villarreal-Rosales
et al. 1992). On the basis of structural grounds and a
feeding experiment showing the incorporation of 14C-

Fig. 5 Characteristic ether lipids of race A (I, III: Metzger and
Casadevall 1991, 1992; II: Metzger 1994)
labelled trienes into the ethers, a biosynthetic pathway was
proposed with two mono-epoxides derived from a C27
triene hydrocarbon as intermediates. In this route, the
protonation of an epoxide function acts as the starter for
the coupling reaction.
Another Bolivian strain (originating also from lake
Overjuyo) is also an efficient producer of an original series
of phenoxy ether lipids accounting for 35% of the dry
biomass (Metzger 1994). Each compound comprises a n-
alkenylresorcinol linked by phenoxy bonds to one or two
hydrocarbon chains derived from alkadienes and/or
alkatetraenes. In some resorcinolic ether lipids, a chain
can be ether-linked to such structures, thus giving rise to
high-molecular-weight lipids C114H206O5; compound II in
Fig. 5). The very low hydrocarbon content of this strain
suggests that alkenylresorcinols, alkadienes and tetraenes,
via their epoxides, are biosynthetic precursors.
Ether lipids occur in all other strains of race A but in a
lower amount than in the three above strains (e.g. 5% dry
weight for a strain originating from Maddingley Brick
Pits, UK; Table 1; Metzger and Largeau 1999). Structu-
rally, they derive from the coupling of alkadienes,
alkenylresorcinols, alkenylhydroquinols and/or α-
branched aldehydes originating from aldol condensation,
called botryals. As an example, Fig. 5 shows an alkenyl-
O-botryalyl ether, compound III, in which a hydroxyl
group is esterified by oleic acid (Metzger and Casadevall
1992). The efficient incorporation of 14C-labelled epox-
yalkene (16%) into ether lipids containing an alkenyl
491
Fig. 6 Characteristic ether lipids of race B (I: Metzger 1999; II:
Metzger et al. 2002; III: Okada et al. 1997b)
moiety, such as III, indicates that epoxide is a close
precursor.
Ethers from B. braunii race B
So far, few ether lipids have been described in B. braunii
race B. Botryolins (Fig. 6) were isolated as very minor
components from a strain originating from Ivory Coast
(Metzger et al. 2002). These triterpenoid triethers comprise
a tetramethylsqualene carbon skeleton. Braunixanthins
(Fig. 6) are carotenoids of a new type isolated from a
Japanese strain (Okada et al. 1997b). They contain an
alkylhydroquinol moiety mid-chain bound by ether bridg-
es to echinenone and a tetramethysqualene derivative. This
latter unit contains a tetrahydrofuran (THF) ring, which
likely derives from the cyclisation a diepoxy-tetramethyls-
qualene (Metzger 1999).
Ethers from B. braunii race L
Twelve series of ether lipids, lycopanerols, were isolated
from B. braunii race L, accounting on the whole for nearly
10% of the dry algal biomass. These compounds present a
great diversity in the arrangement of terpenoid, alkylphe-
nol, resorcinol and/or non-terpenoid units, bound to each
other by ether and/or phenoxy bonds (Rager and Metzger
2000; Metzger and Rager 2002; Metzger et al. 2003). Each
compound contains from one to three tetraterpenoid
moieties exhibiting generally a THF or a tetrahydropyran
(THP) ring, likely derived from diepoxy-lycopane (Fig.
Fig. 7; Metzger 1999). As examples, Fig. 7 shows two
structures of lycopanerols. Lycopanerol F, a C120H236O7
compound, isolated from two strains originating from
India and Ivory Coast, comprises three THF-containing
lycopanes, bound by ether bridges. Lycopanerol H
492
Fig. 7 Characteristic ether li-
pids of race L (I: Metzger 1999;
II: Rager and Metzger 2000; III:
Metzger and Rager 2002)
C147H274O10), also isolated from the same strain of Ivory
Coast, comprises a THF-containing lycopane ether linked
to a THP-containing lycopane, in turn linked by a phenoxy
bond to an alkylhydroquinol linked in the mid-chain to an
α-tocopherol unit. Sequential condensations of terpenoid
and non-terpenoid moieties are believed to be at the origin
of the lycopanerol series (Metzger et al. 2003).
Biotechnological studies
Most of these studies concern strains of race A and, less
frequently, those of race B. Accordingly, the conclusions
cannot be generalised to all B. braunii strains.
In the three chemical races of B. braunii, the hydrocar-
bon productivity was shown to be optimal during the
exponential phase of growth (Largeau et al. 1980; Metzger
et al. 1985a, 1990). Thus, the production of hydrocarbons
appears to be a normal feature of B. braunii. Hydrocarbon
synthesis does not occur in nitrogen- and phosphorous-
deficient media (Casadevall et al. 1985; Brenckman et al.
1985a; Wolf et al. 1985b). Similarly, ether lipid production
was shown to be maximal during the exponential and early
deceleration stages of growth (Villarreal-Rosales et al.
1992).
Like all photosynthetic micro-organisms, B. braunii
requires CO2, light, inorganic nutrients and water to grow.
A modified Chu-13 medium (Largeau et al. 1980; Metzger
et al. 1991), has been found favourable for algae of all
three races. In this culture medium, phosphate concentra-
tion is not a limiting factor for growth (Casadevall et al.
1985). By contrast, an increase in nitrate results in a longer
exponential phase, but in the end this leads to a decrease in
hydrocarbon production, even though the biomass in-
creases. In B. braunii race B, ammonia was found to
inhibit botryococcene biosynthesis, while the synthesis of
some amino acids was favoured (Ohmori et al. 1984). B.
braunii race A is able to use nitrite in the place of nitrate
(Yang et al. 2004a).
By comparison with non-enriched air, air enriched with
1% CO2 highly enhanced growth, with a mean doubling-
time of the biomass in the exponential phase of ca. 2 days
against 7 days and with hydrocarbon production increased
5-fold (Chirac et al. 1985). In contrast, supplementation of
the culture medium with bicarbonate did not decrease the
generation time (Wang et al. 2003).
A wide range of irradiance is accepted by B. braunii:
15–180 W m−2 (Cepák and Lukavsky 1994), but hydro-
carbon synthesis is favoured by a medium light intensity
(40–90 W m−2). In batch air-lift cultures, optimisation of
light intensity leads to a 2-fold increase, both in biomass
and hydrocarbon production (Brenckmann et al. 1985b).
Entrapment of B. braunii colonies in calcium alginate
beads exhibits some interesting advantages by comparison
to free suspension cultures: enhancement in chlorophyll
photosynthetic activity, protection against photoinhibition
towards high irradiance and an increase in hydrocarbon
production despite a decrease in the rate of biomass
production (Baillez et al. 1985, 1986). However, the lack
of stability of calcium alginate beads over a long period is

not favourable to their use in cultures on a large scale.
Immobilization in polyurethane foams appeared detri-
mental, likely due to toxic effects (Baillez et al. 1988).
Although B. braunii forms spectacular blooms in
nature, today its culture in open ponds is far from under
control, essentially due to competition with fast-growing
microalgae. Most studies concerning the influence of
various factors on the production of biomass and
hydrocarbons were carried out in the laboratory using
cylindrical batch air-lift systems (Casadevall et al. 1985;
Kojima and Zhang 1999). Experimental cultures under
natural illumination in tubular photobioreactors, immersed
in water for cooling, have been conducted up to a volume
of 200 l (Gudin and Chaumont 1981, 1984), but their
feasibility at a larger scale has not been tested. Recently,
new forms of photobioreactors, cylindrical and hemi-
spheric, have been developed for culturing microalgae,
including B. braunii (Sato et al. 2002).
The culture of B. braunii on pre-treated domestic and
piggery wastewater reduced both nitrate and phosphate
levels in these media (Sawayama et al. 1994; An et al.
2003). In addition, the concentration of some toxic metals,
such as arsenic, chrome and cadmium, was significantly
reduced (Sawayama et al. 1995). In batch culture, a
biomass of 8.5 g l−1 dry cell weight and 0.95 g l−1
hydrocarbons were obtained from secondarily treated
piggery wastewater (An et al. 2003).
The possibility of using industrial flue gas to supply
cultures with CO2 is supported by the tolerance of B.
braunii towards some pollutants. Indeed, sulfite and
bisulfite (at a low concentration) could supply B. braunii
as sulfur sources, likely via air oxidation into sulfate (Yang
et al. 2004b). Likewise, nitric oxide, via oxidation into
nitrite in water, could be used as the sole source of
nitrogen (Yang et al. 2004a).
Classic routes for the recovery of microalgal biomass,
often dispersed in the culture media, generally need
filtration or centrifugation. In the case of B. braunii race
A, it was shown that adjusting the pH to 11 causes
flocculation of the alga (Lee et al. 1998). Hydrocarbon
recovery can be achieved by extraction of the dry biomass
with solvents (Metzger and Largeau 1999). Supercritical
CO2 extraction has been also employed: the extraction was
found to be optimal at a pressure of 30 MPa (Mendes et al.
2003).
Owing to the localisation of the bulk of hydrocarbons in
the outer walls of the alga (Largeau et al. 1980; Metzger
and Largeau 1999), a short contact of the wet biomass
with a non-toxic solvent was found to be efficient for
extraction (Frenz et al. 1989a, 1989b). By this process, up
to 70% of the total hydrocarbons could be recovered with
hexane as solvent, without reducing cell viability.
Finally, liquid fuel can be derived from B. braunii by
thermochemical liquefaction of the biomass in the
presence of sodium carbonate (Inoue et al. 1994b;
Sawayama et al. 1999).
493
Conclusions
B. braunii is noteworthy for synthesising and accumulat-
ing high amounts of hydrocarbons and ether lipids. The
wide biodiversity of B. braunii results in the production of
three types of hydrocarbons, associated with three chem-
ical races: A (alkadienes, trienes), B (triterpenes) and L
(tetraterpene). In addition, hydrocarbon levels and dis-
tributions vary with algal origin. Botryococcenes and
methylated squalenes, hydrocarbons of race B, have
received much attention due to an original terpenoid
structure, a close biosynthetic relationship with squalene
and an important role in geochemistry. Indeed, the fully
hydrogenated derivatives, botryococcanes and tetrame-
tylsqualane, are specific markers of B. braunii which are
found in petroleum and oil shales, sometimes in high
amounts (Moldowan and Seifert 1980; McKirdy et al.
1986; Summons et al. 2002). Botryococcenes, like meth-
ylated squalenes, originate from the non-mevalonate
terpenoid pathway, likely via presqualene diphosphate as
a close precursor. Successive methylations of C30
botryococcene and squalene give rise to their respective
higher homologues.
In the literature, the oil from B. braunii is often
associated almost exclusively with hydrocarbons. Howev-
er, numerous other compounds, such as ether lipids, are
present in the extracts. These high-molecular-weight ether
lipids derive from lower metabolites, notably hydrocar-
bons, via the coupling of epoxide derivatives. Strains rich
in these unusual compounds could be the source of novel
enzymes responsible for the formation of ether bonds.
Although the influence of various culture parameters on
hydrocarbon production is now well known, cultures on a
large scale of the best candidate species for renewable
hydrocarbons have not yet proved financially worthwhile.
However, the cloning of the squalene synthase gene from a
strain of B. braunii race B in Escherichia coli (Okada et al.
2000) offers promising prospects for the production of
hydrocarbons by fast-growing micro-organisms.
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