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Bioresource Technology 101 (2010) 32743279

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Extraction of hydrocarbons from microalga Botryococcus braunii

with switchable solvents
Chiara Samor *, Cristian Torri, Giulia Samor, Daniele Fabbri, Paola Galletti, Franca Guerrini,
Rossella Pistocchi, Emilio Tagliavini
Interdepartmental Research Centre for Environmental Sciences (CIRSA), University of Bologna, via S. Alberto 163, 48123 Ravenna, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Lipid extraction is a critical step in the development of biofuels from microalgae. Here a new procedure
Received 1 October 2009 was proposed to extract hydrocarbons from dried and water-suspended samples of the microalga Botryo-
Received in revised form 15 December 2009 coccus braunii by using switchable-polarity solvents (SPS) based on 1,8-diazabicyclo-[5.4.0]-undec-7-ene
Accepted 16 December 2009
(DBU) and an alcohol. The high afnity of the non-ionic form of DBU/alcohol SPS towards non-polar com-
Available online 13 January 2010
pounds was exploited to extract hydrocarbons from algae, while the ionic character of the DBU-alkyl car-
bonate form, obtained by the addition of CO2, was used to recover hydrocarbons from the SPS.
DBU/octanol and DBU/ethanol SPS were tested for the extraction efciency of lipids from freeze-dried
Switchable-polarity solvents
B. braunii samples and compared with n-hexane and chloroform/methanol. The DBU/octanol system was
Botryococcus braunii further evaluated for the extraction of hydrocarbons directly from algal culture samples.
Hydrocarbons DBU/octanol exhibited the highest yields of extracted hydrocarbons from both freeze-dried and liquid
Biofuel algal samples (16% and 8.2% respectively against 7.8% and 5.6% with n-hexane).
2009 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2008). For this reason the net energy balance from microalgae
cultivation is still debated (Chisti, 2008a,b; Reijnders, 2008).
The need to replace fossil fuels with fuels derived from renew- Besides the cost of growing and collecting microalgae, down-
able biomass is currently focused on biodiesel from oleaginous stream processes are to be taken into account to evaluate the over-
plant seeds and ethanol from sugarcane/corn, but environmental all productivity. Lipids, which include acylglycerols and
and social concerns are shifting the attention towards the develop- hydrocarbons, represent the most valuable fraction of microalgal
ment of next generation biofuels (Williams et al., 2007). In the case biomass as their high energy content per mass unit is similar to
of terrestrial plant sources, the production of second generation conventional fuels. Several oleaginous microalgae (with lipid con-
biofuels relies on the conversion of the highly abundant and wide- tent exceeding 20% of their dry weight) have been exploited to this
spread non-edible lignocellulosic fraction; a third generation is rep- purpose (Hu et al., 2008), and the biodiesel obtained has been
resented by biofuels from micro and macroalgae. Benets rising claimed to be more convenient than conventional biodiesel from
from the utilisation of aquatic over terrestrial biomass include: (i) plant seeds (Chisti, 2007, 2008a,b).
higher light use efciency (about 5% vs. 1.5%, Posten and Schaub, Lipids can be recovered from algal biomass by means of physi-
2009), (ii) utilisation of marginal areas (e.g. desert and coastal re- cal (cold press) and chemical processes (extraction with solvents)
gions), (iii) possible coupling with other activities (e.g. wastewater or both (Jae-Yon et al., 2010), the latter being more effective; on
treatment, CO2 sequestration), (iv) minor dependence on climatic a laboratory scale, mainly for analytical purposes, the extraction
conditions, (v) availability of a larger number of species and (vi) from freeze-dried samples of algae is usually performed with a
easier genetic manipulation to modify chemical composition (e.g. chloroform/methanol/water mixture (Bligh and Dyer, 1959) and
lipid content) (An et al., 2003; Chisti, 2007, 2008a,b; Rosemberg then the oil is fractionated by column chromatography to obtain
et al., 2008; Shen et al., 2008). However, the industrial development hydrocarbons, non-polar and polar lipids. Non-polar organic sol-
of fuels from microalgae is still hampered by higher overall costs vents such as n-hexane are good alternatives for hydrocarbons
with respect to fossil fuel counterpart: operating open ponds and extraction (Gouveia et al., 2009). However, in the last years differ-
bioreactors are expensive and the harvesting of algal biomass is en- ent efforts have been done to reduce the use of toxic and polluting
ergy costly (Benemann, 1997; Molina Grima et al., 2003; Wang organic solvents to improve the sustainability of the extraction
procedures, for example by using supercritical uids (Aresta
* Corresponding author. Tel.: +39 0544 937353; fax: +39 0544 937411. et al., 2005; Mendes et al., 2003). In this eld, switchable solvents
E-mail address: (C. Samor). could represent a new fascinating class of extracting media, as they

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
C. Samor et al. / Bioresource Technology 101 (2010) 32743279 3275

are capable to turn from a non-ionic form to an ionic liquid by sim- The alga was grown in a modied Chu13 medium (Largeau
ply bubbling CO2, and to be reconverted in the non-ionic form by et al., 1980) containing the following components (g/l): KNO3
bubbling N2 (Jessop et al., 2005). In particular, switchable-polarity (0.2), K2HOP4 (0.04), MgSO47H2O (0.1), CaCl26H2O (0.08), Fe cit-
solvents (SPS) exhibit two degrees of polarity: lipophylic in the rate (0.01), citric acid (0.1); micro elements: B, Mn (both at
non-ionic form and hydrophilic in the ionic one, ranging from 0.5 ppm), Zn (0.05 ppm), Cu, Co, Mo (0.02 ppm). The pH was ad-
the polarity of chloroform to that of DMF; this feature can be justed to 7.5 with KOH before autoclaving. Culture vessel (2 l
exploited in many processes, such as extractions (Blasucci et al., Erlenmeyer ask) was aerated and thermostatically controlled at
2009; Phan et al., 2009, 2008a.) as well as chemical synthesis, to 20 C in a shaking incubator under 120 lEm2 s1 light intensity
avoid the use and the waste of solvents of different polarity for with 16-h light: 8-h dark cycle. The aliquot of the culture used
each process step (Donaldson et al., 2009; Phan et al., 2008b). for the extractions was routinely harvested, by centrifugation, at
The synthesis of new switchable solvents is increasing rapidly, the beginning of the stationary phase.
according to the needs of systems with specic physic-chemical The dry weight of algal biomass was determined gravimetri-
properties (Yamada et al., 2007); the most known SPS, the DBU-al- cally; aliquots of 10 ml algal suspension were ltered through a
kyl carbonate salts, are usually obtained from a 1:1 mixtures of pre-weighted, pre-combusted (60 C, 24 h) glass bre lter (What-
two neutral liquid components, 1,8-diazabicyclo-[5.4.0]-undec-7- man GF/F, 47 mm, nominal pore size 0.7 lm). The lters were then
ene (DBU) and an alcohol whose chain length can be chosen to - dried at 60 C to a constant weight.
nely tune the polarity of the non-ionic form and the melting point
of the ionic form (Phan et al., 2008b). 2.3. Standard compounds
Despite the increasing interest towards SPS, there is only one
study in the literature about their potential use as extracting media Calibration solutions for GCMS analysis of hydrocarbons were
of lipids from biomass useful for energetic applications. The pro- composed by the four main long chain alkenes C27H52, C29H56,
cess described by Phan et al. (2009) developed a new application C29H54 and C31H60 isolated from B. braunii samples. In a typical pro-
of DBU/ethanol to natural matrices, to extract soy oil from soybean cedure (Yamaguchi et al., 1987 with slight modications), 100 mg
akes, facing the problem of the large amount of n-hexane re- of freeze-dried alga were extracted twice with n-hexane at 60 C
quired for the industrial process. To the best of our knowledge, (2 ml and 2 h each time) and ltered; the n-hexane solutions, con-
there are no studies on the performance of SPS as extracting sys- taining the hydrocarbons, were collected, evaporated under vac-
tems for lipids from algal biomass. uum, puried by chromatographic column and used to determine
In this work, the SPS systems based on DBU have been investi- the GCMS response factor relative to hexatriacontane (Sigma
gated to extract hydrocarbons from Botryococcus braunii. Aldrich).
B. braunii is a freshwater colonial green microalga capable to Tridecanoic acid was utilised as internal standard to quantify
produce high levels of liquid hydrocarbons. There are three main free fatty acids and total fatty acids, sum of free fatty acids and
B. braunii races, each one synthesising different types of olenic the bounded fatty acids (e.g. triglycerides), assuming an unitary re-
hydrocarbons: the A race accumulates linear olens, odd num- sponse factor relative to tridecanoic acid.
bered from C23 to C31, chiey C27, C29, and C31 dienes or trienes;
the B race produces polyunsaturated triterpenes (botryococcenes),
while the L race synthesises one single tetraterpenoid hydrocarbon 2.4. Extraction of lipids from freeze-dried algae
named lycopadiene (Metzger et al., 1985a,b). Specically for B.
braunii the bulk of hydrocarbons is located in an external cellular Samples of freeze-dried alga (30 mg) were extracted at 60 C for
pool and it can be recovered after a short extraction with n-hexane 4 h with each of the following solvent systems: (i) n-hexane (4 ml)
(Largeau et al., 1980). followed by a mixture 2/1 of chloroform/methanol (4 ml), (ii) DBU
The aim of this work was to compare the lipid extraction ef- (3 ml), (iii) equimolar mixtures of DBU/ethanol (6.6 mmol, 1 ml/
ciency of two SPS systems based on DBU/ethanol and DBU/octanol 0.388 ml), (iv) or DBU/octanol (6.6 mmol, 1 ml/1 ml).
with that of traditional solvents (n-hexane, chloroform/methanol) DBU alone was tested as extractive solvent in order to verify if
from freeze-dried and liquid samples of B. braunii A race. In partic- the addition of octanol to achieve the SPS system, before or after
ular we have exploited the lipophylicity of the non-ionic form of the extraction process could affect in some way the efciency of
SPS (Jessop et al., 2005) to extract algal hydrocarbons, which are the process itself.
then recovered from the SPS solution by a simple bubbling of After cooling, an aliquot of each extract was spiked with inter-
CO2, using the low afnity of the ionic form for these molecules. nal standards (hexatriacontane and tridecanoic acid), diluted with
n-hexane and analysed by GCMS to determine the quantity of ex-
tracted hydrocarbons and fatty acids. In the case of DBU/ethanol
2. Methods and DBU/octanol the extraction time was evaluated by GCMS
analysis at increasing time intervals.
2.1. Chemicals
2.5. Extraction of lipids from algal cultures
DBU (Aldrich, 98% grade) was distilled (b.p. = 82 C, 0.8 mbar)
and stored under N2. All other solvents and chemicals were used
An aliquot (3 ml) was withdrawn from cultures at concentra-
as received from Aldrich.
tion of 0.8 g algal dry weight/l and added to the extraction medium
Supercritical grade CO2 (99.95%, H2O < 20 ppm), nitrogen
in a centrifuge tube, without stirring and at room temperature. The
(99.998%, H2O < 3 ppm) were used as received from Air Liquide.
biphasic system was then centrifuged at 300 rpm for 2 or 24 h, or
at 3000 rpm for 4 h. The following extracting media were tested:
2.2. Microorganism and culture conditions (i) n-hexane (3 ml), (ii) octanol (3 ml), (iii) equimolar mixture of
DBU/octanol (9.9 mmol, 1.5 ml/1.5 ml). In the latter case, the pH
B. braunii A race (SAG 807-1) was obtained from the Algal Cul- of the aqueous phase was brought to 13 with KOH, to allow the
ture Centre, University of Gttingen, Germany. SAG 807-1 was iso- complete separation of DBU from water.
lated from a water sample collected in Madingly Brick Pits, Octanol alone was tested as extractive solvent in order to verify
Cambridge, by M.R. Droop in 1950. if the addition of DBU to achieve the SPS system, before or after the
3276 C. Samor et al. / Bioresource Technology 101 (2010) 32743279

extraction process could affect in some way the efciency of the ltration in the case of cultures. Reported yields were averaged
process itself. from three replicate extractions.
Centrifuging was chosen to obtain a clearer separation (useful
for small samples operation) of the aqueous and organic phases.
This method allows to avoid vigorous stirring that should end up 3. Results and discussion
into an untreatable foaming.
The upper organic phase was collected and dried at room tem- 3.1. Extraction of lipids from freeze-dried B. braunii samples
perature under N2 (1 l min1) for about 30 min, until the water
content was less than 0.1% by Karl Fischer titration. The excellent afnity of switchable systems for non-polar com-
An aliquot of the upper organic phase was spiked with hexatri- pounds was exploited to extract the hydrocarbons produced in
acontane as internal standard, diluted with n-hexane and analysed high amount by B. braunii. The choice of the alcohol is a critical
by GCMS to determine the quantity of extracted hydrocarbons. point to form the proper liquid carbonate anion. Previous works
(Heldebrant et al., 2005; Munshi et al., 2002; Prez et al., 2004)
demonstrated that bicarbonate and methyl carbonate DBU salts
2.6. Recovery of hydrocarbons with DBU/octanol system
have melting points lower than room temperature; but DBU salts
with longer alkyl chains are liquid at room temperature (Phan
After the extraction of freeze-dried algae with DBU/octanol was
et al., 2008b), therefore suitable for the extraction process. For this
completed and analysed by GCMS as described in Section 2.4, the
reason, we selected DBU-ethyl carbonate and DBU-octyl carbonate
algal biomass was ltered off and CO2 was bubbled into the stirred
which have melting points of 35 C and 30 C respectively (Phan
solution at 40 C for 1 h. Upon formation of DBU-octyl carbonate
et al., 2008b).
salt, an hydrocarbon layer separated at the top of the ionic liquid
The rationale of the proposed process for the extraction of
phase. n-Hexane was added to the surface layer without mixing,
freeze-dried algal samples is described in Fig. 1.
and the resulting hexane solution was decanted and subjected to
The performance of the extraction of DBU systems was
GCMS analysis, to verify the degree of contaminations with DBU
and octanol of the algal oil. Then, the DBU-octyl carbonate mixture
The yields of total hydrocarbons and of the ve major linear ole-
was heated at 60 C, while N2 bubbled through the solution, to con-
ns (C27H52, C29H56, C29H54 and C31H60) obtained from freeze-dried
vert back the system into the non-ionic form, which was subjected
samples using different solvents are shown in Fig. 2. The best re-
to GCMS analysis, to verify the degree of hydrocarbon recovery.
sults were obtained with the equimolar mixture DBU/octanol
A procedural blank was investigated by NMR: 1H and 13C NMR
(16 2% total hydrocarbons yield), followed by DBU alone
spectra were collected in CDCl3 on a VARIAN Mercury 400 spec-
(15 6% yield), and by DBU/ethanol (12 2% yield). The two SPS
trometer; peaks corresponding to those of DBU-octyl carbonate
were both better than n-hexane, that afforded the lowest yields
(after CO2 treatment) and equimolar DBU/octanol mixture (after
(7.8 3%), after an extraction process performed at the same tem-
N2 bubbling) reported by Phan et al., 2008b were only observed.
peratures and after the same time interval (60 C and 4 h).
As expected, all the tested solvents showed no selectivity
2.7. GCMS analysis among different olens.
The yields relative to fatty acids, presented in Fig. 3, were rather
Hydrocarbons were analysed without any pre-treatment. The low with DBU (0.6 0.2%) as well as with the two switchable mix-
amount of free fatty acids was obtained by silylation and GCMS tures (0.7 0.1% DBU/octanol and 0.6 0.2% DBU/ethanol). These
analysis. Five milligrams of the extract were spiked with 100 ll extracting media were less efcient than a n-hexane/chloroform/
of internal standard solution, 200 ll of bis-trimethylsilyl-tri- methanol mixture (2.7 0.3% yield). No selectivity towards a spe-
uoro-acetamide containing 1% trimethyl-chlorosilane and 10 ll cic fatty acid was observed in any case.
of pyridine. We evaluated also if the presence of free fatty acids in the algal
The amount of total fatty acids (sum of free and bounded fatty oil could affect the separation process; this could be a problem for
acids) was obtained by transesterication into the corresponding the extraction of biomass with a high content of free fatty acids. In
methyl esters (FAMEs). The extract was reuxed for 10 min with the case of the microalga B. braunii A strain, we found a percentage
4 ml methanolic NaOH 0.5 M; then 5 ml of MeOH/BF3 were added of free fatty acids of 0.6% on a dry weight basis (according to data of
and the mixture reuxed for 2 min followed by the addition of Yamaguchi et al., 1987) that was irrelevant if compared with the
10 ml of n-hexane. After cooling, a saturated NaCl aqueous solution amount of DBU used for the extraction which could eventually re-
was added under stirring, then the hexane layer containing FAMEs act with the fatty acids (about 0.18 mg of free fatty acids respect to
was separated, dried over anhydrous sodium sulphate and diluted 1 g of DBU).
prior to GCMS analysis (Fabbri et al., 2005). The effect of alcohol chain length on the rate of extraction was
Analyses were performed by a 6850 Agilent HP gas chromato- investigated by determining the extraction yields at selected time
graph connected to a 5975 Agilent HP quadrupole mass spectrom- intervals (Fig. 4). DBU/octanol and DBU/ethanol exhibited approx-
eter. Analytes were separated by a HP-5 fused-silica capillary imately the same behaviour, with similar hydrocarbons amount
column (stationary phase poly[5% diphenyl/95% dimethyl]siloxane, extracted after 240 min (14% and 13% yields respectively).
30 m, 0.25 mm i.d., 0.25 mm lm thickness) using helium as carrier
gas (at constant pressure, 33 cm s1 linear velocity at 200 C). Mea-
surement solutions were injected under splitless condition with 3.2. Extraction of lipids from B. braunii cultures
injector temperature set at 280 C. Mass spectra were recorded un-
der electron ionization (70 eV) at a frequency of 1 scan s1 within The direct extraction from algal cultures was performed with n-
the 12450 m/z range. The following thermal program was used: hexane, octanol and equimolar DBU/octanol mixture; in each
130 C for 3 min, then 10 C/min up to 300 C and hold for 10 min. extraction system, the solvent layer was stratied over the water
The quantity of extracted compounds was reported as percent- phase, avoiding the direct contact of the extraction agent with
age yields of the extracted algal biomass. This latter gure was cal- the algal cells. The extraction with DBU/octanol from cultures re-
culated on a dry weight basis in the case of freeze-dried samples, quired a previous adjustment of the pH to alkaline conditions to
and on the algal biomass content of the liquid samples following achieve phase separation.
C. Samor et al. / Bioresource Technology 101 (2010) 32743279 3277

N +
alga alga

N + OH
N +


N 7 O O
N 7

Fig. 1. Strategy for the extraction of hydrocarbons from freeze-dried samples of B. braunii by means of switchable-polarity solvents exemplied for DBU/octanol.




C27H52 C29H56 C29H54 C29H54 C31H60 TOTAL

Fig. 2. Hydrocarbon yields calculated on a dry weight basis, obtained with n-hexane ( ), DBU ( ), equimolar mixtures of DBU/octanol (h), and DBU/ethanol ( ), from freeze-
dried samples (60 C, 4 h).





fatty acids

Fig. 3. Fatty acids yields calculated on a dry weight basis, obtained with n-hexane/chloroform/methanol ( ), DBU ( ), equimolar mixtures of DBU/octanol (h), and DBU/
ethanol ( ), from freeze-dried samples (60 C, 4 h).

The extractions were performed at room temperature, stirring In Fig. 5 the extraction yields after 2 h and 24 h of centrifugation
samples in centrifuge at different speeds, simulating a high at 300 rpm, and after 4 h at 3000 rpm, are shown. Although the
(3000 rpm) and a low energy (300 rpm) liquid/liquid extraction extraction process is somewhat sluggish, the equimolar mixture
process from the growth medium. DBU/octanol after 24 h at 300 rpm gave 8.2% hydrocarbon yield,
3278 C. Samor et al. / Bioresource Technology 101 (2010) 32743279



total hydrocarbons


0 50 100 150 200 250

Fig. 4. Hydrocarbons extraction efciency of freeze-dried algal samples with time using equimolar mixtures of DBU/octanol (4), and DBU/ethanol (d). Dots and dashes curve,
and continuous line represent the logarithmic regression of DBU/octanol and DBU/ethanol data respectively.

total hydrocarbons


octanol hexane DBU/octanol

Fig. 5. Hydrocarbon extraction efciency of culture medium suspended algal samples calculated on a dry weight basis, with octanol, n-hexane, and equimolar mixture of
DBU/octanol, after 2 ( ), and 24 h ( ) centrifugation at 300 rpm, and after 4 h ( ) centrifugation at 3000 rpm.

slightly higher than that obtainable from freeze-dried sample with equimolar mixtures of DBU/octanol used for the extraction of
n-hexane under reux (7.8% yield). freeze-dried algal samples, and then by converting the DBU-octyl
Similarly to what observed with dried samples, the best extrac- carbonate salt into the non-polar form by bubbling N2. The ef-
tive solvent for liquid samples was the switchable mixture DBU/ ciency of a non-ionic/ionic cycle in recovering pure hydrocarbons
octanol (8.2 1% yield), followed by n-hexane and octanol with was about 81% of the total amount of hydrocarbons extracted, with
5.6 2% and 4.4 0.4% yield respectively, conrming a positive role 8.1% of hydrocarbons retained in the ionic SPS phase in the second
of DBU in the extraction process when mixed with octanol. half of the cycle, and about a 10% mechanical loss probably due to
The effect of the centrifugation speed was also evaluated: for all the small size of the sample. The results indicated that the ionic
the tested solvents, at higher rate (3000 rpm) the extraction re- form retained a rather small amount of the hydrocarbons extracted
sulted faster, obtaining in 4 h approximately the same yields with the equimolar mixture DBU/octanol. This amount could be
allowing to obtain at 300 rpm for 24 h (DBU/octanol 7.0 0.9% still reduced by increasing the size of the extracted sample.
yield, n-hexane 5.1 0.7% and 4.4 0.5% in octanol). This could The GCMS analysis of the hydrocarbons obtained after the pro-
be explained by the fact that by raising the centrifuge rate less cedure described above indicated that the oil still contained small
dense algae (with higher hydrocarbon content) will move quickly amounts of octanol (0.3%) and DBU (0.4%); bubbling extra CO2 for 1
to the top of the water phase and release the hydrocarbons in h at 40 C decreased the levels of contamination to undetectable
the upper organic layer by contact. values because of the precipitation of all the ionic liquid from the
The water content in the upper organic phase, after the adjust- oil.
ing of the pH of the aqueous phase with KOH, was 7% (g H2O/
100 ml SPS, Karl Fischer titration). To avoid the formation of
DBU-bicarbonate, a further step to remove the residual water 4. Conclusions
was necessary before treating the SPS with CO2. Water was re-
moved from DBU/octanol by bubbling N2 for 30 min at room tem- The potential role of SPS as a green technology for the extrac-
perature; after this time the residual H2O in the system was less tion of hydrocarbons from both dried microalgal biomass and di-
than 0.1%, without any loss of the organic components. rectly from aqueous growth medium was demonstrated. The
DBU/octanol system exhibited a better extraction efciency than
3.3. Recovery of hydrocarbons with DBU/octanol system conventional solvents, both with dried and liquid samples. This is
an important issue considering that the harvest and the dewater-
A complete non-ionic/ionic cycle to recover the extracted ing of algal biomass have a large impact on overall costs and en-
hydrocarbons was performed rst by bubbling CO2 into a stirring ergy balance.
C. Samor et al. / Bioresource Technology 101 (2010) 32743279 3279

Besides the efciency in the hydrocarbons extraction, SPS have Jae-Yon, L., Chan, Y., So-Young, J., Chi-Yong, A., Hee-Mock, O., 2010. Comparison of
several methods for effective lipid extraction from microalgae. Bioresource
the advantage to be recyclable non-volatile/non-inammable sys-
Technol. 101, S75S77.
tems, therefore suited for non-hazardous small plants for biofuel Jessop, P.G., Heldebrant, D.J., Li, X., Eckert, C.A., Liotta, C.L., 2005. Reversible non
production located nearby algal cultivation sites. polar-to-polar solvent. Nature 436, 1102.
Largeau, C., Casadevall, E., Berkaloff, C., Dhmelincourt, P., 1980. Sites of
accumulation and composition of hydrocarbons in Botryococcus braunii.
Acknowledgements Phytochemistry 19, 10431051.
Mendes, R.L., Nobre, B.P., Cardoso, M.T., Pereira, A.P., Palavra, A.F., 2003.
Supercritical carbon dioxide extraction of compounds with pharmaceutical
We acknowledge the ministry MiUR and the University of Bolo- importance from microalgae. Inorg. Chim. Acta. 356, 328334.
gna (RFO programme) for funding. Metzger, P., Casadevall, E., Pouet, M.J., Pouet, Y., 1985a. Structures of some
botryococcenes: branched hydrocarbons from the b-race of the green alga
Botryococcus braunii. Phytochemistry 24, 29953002.
Metzger, P., Berkaloff, C., Casadevall, E., Coute, A., 1985b. Alkadiene- and
Appendix A. Supplementary data botryococcene-producing races of wild strains of Botryococcus braunii.
Phytochemistry 24, 23052312.
Molina Grima, E., Belarbi, E.H., Acin Fernndez, F.G., Robles Medina, A., Chisti, Y.,
Supplementary data associated with this article can be found, in
2003. Recovery of microalgal biomass and metabolites: process options and
the online version, at doi:10.1016/j.biortech.2009.12.068. economics. Biotechnol. Adv. 20, 491515.
Munshi, P., Main, A.D., Linehan, J., Tai, C.C., Jessop, P.G., 2002. Hydrogenation of
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