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Renewable and Sustainable Energy Reviews 64 (2016) 260270

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Renewable and Sustainable Energy Reviews

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Botryococcus braunii for biodiesel production

Marija B. Tasi a,n,1, Luisa Fernanda Rios Pinto b, Bruno Colling Klein b,c, Vlada B. Veljkovi a,
Rubens Maciel Filho b
Faculty of Technology, University of Ni, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia
State University of Campinas, UNICAMP, Laboratory of Optimization, Design and Advanced Control, Department of Chemical Process, School of Chemical
Engineering, Av. Albert Einstein, 500 CEP 13083-970, Campinas, Sao Paulo, Brazil
CNPEM/CTBE, Brazilian Bioethanol Science and Technology Laboratory, Division of Integrated Assessment of Bioreneries, Rua Giuseppe Mximo Scolfaro,
10000, Campinas, Sao Paulo, Brazil

art ic l e i nf o a b s t r a c t

Article history: This paper presents a review on microalga Botryococcus braunii (B. braunii) with an emphasis on lipid
Received 22 July 2015 production for possible industrial commercialization in biodiesel production. Summarizing the nature of
Received in revised form this alga is fundamental to establish the detail environment conditions for optimal biomass growth and
19 April 2016
the production of lipids, as well as upstream processing technologies for biodiesel production. This study
Accepted 15 June 2016
focuses the attention on identifying possible avenues for further investigation in the design of cultivation
systems and the integration of process technologies at industrial scales. The advantages and dis-
Keywords: advantages of biodiesel production processes based on these algal lipids are also analyzed. A particularly
Botryococcus braunii promising process involving B. braunii combines ue gas and wastewaters to produce lipids, converting
them to biodiesel by supercritical (SC) direct trans/esterication (esterication followed by transester-
ication). We conclude that such processes using B. braunii have considerable potential for the pro-
Harvesting duction of cleaner and more efcient biodiesel than current methods, reducing the environmental im-
Trans/esterication pact of fossil fuel use.
& 2016 Elsevier Ltd. All rights reserved.


1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
2. B. braunii as a biodiesel feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
3. Toxic blooms and competition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
4. Growth environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
5. Biodiesel technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
5.1. Upstream pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
5.1.1. Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
5.1.2. Harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
5.1.3. Dewatering of harvested biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
5.1.4. Lipid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
5.2. Trans/esterication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
5.3. Downstream purication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
6. The quality of biodiesel obtained from B. braunii's lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

Corresponding author.
E-mail addresses: marijat14@yahoo.com, mtasic@tf.ni.ac.rs (M.B. Tasi).
Present address: State University of Campinas, UNICAMP, Department of Process Development and Products, Av. Albert Einstein, 500 CEP 13083-970, Campinas, Sao
Paulo, Brazil.

1364-0321/& 2016 Elsevier Ltd. All rights reserved.
M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270 261

7. Economic assessment and life cycle analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

8. Conclusion and scope for future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

1. Introduction braunii biodiesel production to identify technologies with im-

proved performance indicators. First of all, B. braunii is considered
The high carbon dioxide (CO2) concentration in the atmo- as a biodiesel feedstock referring to its nature, cultivation (in-
sphere, mainly the result of fossil fuel combustion, is thought to be cluding contamination problems and nutritional requirements),
one of the primary causes of global warming [1]. Biodiesel, being a harvesting, lipid extraction, and treatments to obtain FAMEs. Then,
renewable fuel, presents a promising alternative solution over biodiesel production processes based on these algal lipids are
fossil diesel. Biodiesel is typically generated by chemical reactions analyzed, highlighting both their advantages and disadvantages. It
(commonly known as trans/esterication) between lipids (e.g., is worth to emphasize that while relevant and readable reviews
triacylglycerols, abbreviated TAGs), free fatty acids (FFAs), and al- have been published on the biodiesel production from various
cohol (commonly methanol or ethanol), which generate fatty acid algal species [1114], none has included B. braunii.
alkyl esters. A variety of lipid-rich feedstocks can be used to pro-
duce biodiesel. These include: naturally derived crops; rened,
used or waste vegetable oils; animal fats; and algae lipids or 2. B. braunii as a biodiesel feedstock
sewage sludge. Algae are a particularly promising lipid source due
to their high lipid content and possibility to x atmospheric CO2 B. braunii is a green, pyriform shaped planktonic microalga,
through photosynthetic capture. They can also be cultivated on with colonies growing in the cluster and that can be found in
non-agricultural land or in aquatic environments, using waste- temperate or tropical oligotrophic lakes and estuaries, widespread
water as a nutrient source and without competing with food in freshwater and brackish lakes, reservoirs, ponds, or even
production or impacting land use. ephemeral lakes. B. braunii strains can be found in all climate
Algal biodiesel industrial production is still far from being zones except Antarctica [8]. Cell size (length  width) is in the
commercially viable, but it appears to be potentially suitable [2,3]. range of (89  5) mm to (13  79) mm [15]. Among various micro
Current problems include: (i) the considerable usage of non-agri- and macro algae species, B. braunii has been identied as the most
cultural land, fertilizers and water; (ii) high energy demands; (iii) promising for biofuel production, because of its excellent lipid
the relatively high up-front capital costs and the high price of production capability (content up to 65%, see Table 1). Although
biodiesel; and (iv) the poor transportation, storage, combustion, this microalga is known for high amounts of hydrocarbons and
and drivability performance of biodiesel. Several studies have ether lipids [16,17], its high content of saturated and mono-
pointed out that fatty acid methyl esters (FAME) present a variety unsaturated FAs as well as TAGs [18,19] makes it suitable for bio-
of problems when applied to automobile fuels [4]. However, the diesel production. The percentages of saturated, monounsaturated
efciency and quality of biodiesel production are heavily inu- and polyunsaturated FAs in B. braunii dry biomass revolve around
enced by the lipid extraction and lipid conversion methods used 9.85%, 79.61% and 10.54%, respectively [10]. Most of the FAs are
[57], and so the choice of feedstock is vital. The development of synthesized during the steady-state phase of algae growth, with
energy efcient and protable technologies through process in- the highest content of palmitic and oleic acids located in-
tegration is, therefore, a high priority for algal biodiesel tracellularly, but also in small amounts in the extracellular
production. medium.
The lipid content, the fatty acids (FAs) composition of TAGs and Lipid secretion in the extracellular medium is an unusual
quality (for example, iodine value (IV), cetane number (CN), FFAs phenomenon of B. braunii because all known species of microalgae
content) are the most important factors for selecting the right have cytoplasmic lipids. For example, the FAs lipids biosynthesis
species of microalgae. Polar and neutral lipids are preferred for the by B. braunii begins in the plastid, elongates within the en-
biodiesel production. Phaeodactylum tricornutum, Chlorella vul- doplasmic reticulum, transfers to the plasma membrane of the
garis, Chlorella protothecoides, B. braunii, and Nannochloropsis sali- endoplasmic reticulum or via Golgi bodies and then secretes to the
na are identied to produce such lipids [8]. Among all mentioned cell surface [46]. Lipid droplets are seen on the surface of the cell
species, B. braunii emerges as a promising lipid source because of apex [46]. The lipid yield and composition vary with the strain,
its widespread occurrence in aquatic environments and high lipid growth conditions and cell aging [4749].
content. According to an Aquatic Species Program (ASP) report [9],
B. braunii would not function well as a feedstock for the lipid-
based fuel production due to its slow growth (doubling time of 3. Toxic blooms and competition
72 h). The ASP also found this alga lipid to be less suitable for
transesterication (i.e. creating biodiesel), having most of its lipids The colonial B. braunii is a bloom-forming alga. The cause of the
as C29 to C34 aliphatic hydrocarbons, and less abundance of TAGs blooms and their subsequent damage to the populations of other
and C18 FAs. The FA proling of B. braunii (IBL-C117) is not ap- organisms has been widely studied [50,51]. Blooms are char-
propriate for producing a pure American Society of Testing and acterized with a high lipid content, about 40% of the dry biomass,
Materials (ASTM) nor the Brazilian Agency for Fuels and Biofuels compared with 18% for the culture collection strain [18]. Further-
(Agncia Nacional do Petrleo, Gs Natural e Biocombustveis, more, the lipid composition of bloom strains is represented mostly
ANP) grade biodiesel [10]. However, newer research shows that by saturated and monoenoic FAs. The dominant FAs are palmitic,
the doubling time could be reduced to 48 h and that both algal oleic, and linolenic acids. These FAs allow B. braunii to dominate in
lipid composition and FA proling are strongly affected by its en- natural environments due to their toxic effects on sh and zoo-
vironment growth conditions. plankton. Nonetheless, B. braunii's blooming nature is advanta-
The aim of this paper is to obtain a comprehensive picture of B. geous for biodiesel production due to quantity and quality of the
262 M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270

Table 1
B. braunii strains of importance for biodiesel production.

Culture collection Strain Biomass Lipid contenta References

UTEX, USA LB-572 (96.47 6.8) g/Lb (26.80 72.05)% [20]

(0.0150.02) g/(L day)c 15.6%c [21]
0.304 g/(L day) 64.96% [22]
LB-572, A race 1.5 g/L 28% [23]
1.8 g/L 24%d [24]
UTEX 572 0.38 g/L 63% [25]
1 g/L 34.6% [26]
Austin 0.035 g/(L day) (46.85 75.20)% [27]
(0.026.55 7 7.66) (g/L d) 24% [28]
Institute of Hydrobiology, Chinese Academy of Sciences, PR China FACHB 357 5.5 g/(m2 d) 42% [29]
SAG culture collection, University of Gttingen, Germany SAG 807-1 6.45 g/(m2 d) 3.4412.44%e [30]
Gttingen 807/l Droop 0.032 g/(L day) (21.747 3.94)% [27]
Institute of Process Engineering, Chinese Academy of Sciences IPE 001 0.043.88 g/(L day) 46.39% [31]
Institute of Plant Physiology of the Russian Academy of Sciences Ktz No LB 807/1 Droop (0.16 70.01) g/(L d) 18.0% [18]
1950 H-252
1517%f [19]
0.7 g/L 22% [32]
Lake Shira (Khakasia, Siberia) A bloom of B. braunii, A g 40% [18,33,34]
Microalgal Culture Collection of the K.A. Timiryazev Institute of Ktz IPPAS H-252h,i 3.5 g/L 21% [35]
Plant Physiology RAS (IPPAS)
1.15 g/L (19.9 7 1.4)% [36]
Freshwater lake Kolleru, Andhra Pradesh, India Ktz. AP103j (1.7 70.051) (1.8 7 13) g/L 177 0.87 (19 7 0.98)% [37]
Ktz. AP104 (1.28 70.038) g/L (33079.9) mg/L
Ktz. AP105 (1.167 0.035) g/L (320 7 9.6) mg/L
Centre for Advanced Studies in Botany, University of Madras, Chen- AP102 1.9 g/L 48.40% [38]
nai, India
Natural Brazilian ecosystems LEM 14 1.88 g/L 36.14% [39]
Biogeochemistry and Ecology of Terrestrial Environments Depart- A race 0.46 g/(L day) 17.85% [40]
ment at the cole Nationale Superieure de Chimie de Paris
Lake of Coat at Herno, Brittany A race 60.6% [41]
UTEX, USA Austin A race 27.4 g/100 g [42]
Fresh water UTEX 572 (0.037 70.005) (g/L d) (13.50 7 3.78)% [43]
Lake in Trag province, Thailand TRG 5.16 g/L 37.5% [44]
Freshwater reservoir in Assam, India GUBIOTJTBB1 0.316 g/L (57.147 1.18)% [45]

Total lipids.
Immobilized system cultivation.
Outdoor production under uncontrolled conditions.
Intracellular lipids. The content of total extracellular lipids is not available.
Non-polar lipids.
Raceway pond cultivation.
Not available.
Due to hydrocarbon and fatty acid composition this strain is closer to Botryococcuss udeticus Lemmermann.
Also known as B. braunii Ktz No LB 807/1 Droop 1950 from Culture Collection of Algae and Protozoa, Dunstaffnage, Scotland.
Open raceway pond conditions.

synthesized lipids. 4. Growth environment

Because of the relatively slow growth of B. braunii, trials to
cultivate B. braunii in non-axenic systems often fail due to biomass The major target in industrial B. braunii biodiesel production is
contamination from other green algal and cyanobacterial species. increasing biomass production while improving the quantity and
B. braunii's competitors include Chlorella vulgaris, Chlamydomonas quality of the lipid product in a sustainable way. Carbon, nitrogen,
reinhardtii, Scenedesmus dimorphus, Pseudokirchneriella sub- phosphorus, sulfur, and iron are essential nutrients both for bio-
capitata, and Merismopedia tenuissima [52]. Contamination among mass productivity and for the quantity and quality of the synthe-
algal species is a consequence of competition among various or- sized lipids. To reach this target, many synthetic media have been
investigated [20,23,24,28,29,39,5560], but only the modied
ganisms for the critical nutrient in the medium. Furthermore, this
Chu13 medium [29,30,61] has been shown to be optimal. How-
contest is mainly inuenced by the biomass ratio of competing
ever, commercial biodiesel production using this synthetic med-
species. For example, the low or medium ltrate density of two
ium is mostly discouraged, as the high consumption rates of fossil-
common algae species in natural waters - Chlorella vulgaris and
based fertilizers as the source of inorganic nutrients make it costly
Chlamydomonas reinhardtii - stimulates B. braunii growth, while
and unsustainable. It means that the modied Chu13 medium is of
high cell ltrate density cultures signicantly inhibit it [53]. The
importance only for B. braunii inoculum preparation at commercial
presence of bacteria is also associated with the mass culture of B. scales.
braunii. For example, the gram-negative Rhizobium sp. [54] forms a Recent research has been directed towards the cultivation of B.
biolm (microalgal matrix) and not only signicantly stimulates B. braunii in natural media. For example, since the natural habitat of
braunii growth but also produces the cell-to-cell signal molecules B. braunii is seawater, recently published papers suggest the pos-
known as acyl-homoserine lactones. The formation of B. braunii's sible exploitation of NaCl-rich seawater for the outdoor commer-
bloom is also one of the outcomes for contamination caused by cial cultivation of B. braunii [62,63]. However, not all B. braunii
other microorganisms. strains are halophilic. The biomass yield and the lipid content of B.
M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270 263

braunii (LB 572) increase under all tested NaCl concentrations concentration (0.42 mM). It was explained by the fact that the B.
(0.0170.085 M), reaching the highest content of palmitic and oleic braunii (KMITL 2) strain was not pre-cultured before medium in-
acid at 0.085 M [23]. Higher concentrations of NaCI (0.50.75 M) oculation, which would have helped the algal cells to reproduce
are either toxic to the Austin and Gottingen strains, respectively further.
[27] or decrease the biomass concentration and the lipid yield of B. Phosphorus and sulfur concentrations also have major impacts
braunii KMITL 2 [64]. Zhila et al. [36] show that haloadaptation is a on both the growth and lipid productivity of B. braunii (LB 572), as
specicity of the strain growth phase. For example, the presence of demonstrated by statistical optimization of culture media [56].
salt (0.3 M and 0.7 M) in the medium inhibits the growth of B. Phosphorus - usually in the form of phosphate - at concentrations
braunii Ktz (IPPAS H-252), enriching TAGs and oleic acid content up to 444 mg/L increased the biomass concentration and reduced
for the rst three days, and increasing biomass and polyenoic FAs the lipid content 7.3 fold and 1.7 fold, respectively [64]. Lipid
content in later growth phases [36]. The cultivation of B. braunii in productivity also increased 1.2 fold by enhancing the phosphate
seawater shows promising results in reducing the CO2 content concentration from 40 mg/L to 83 mg/L [56]. However, it should be
since xed atmospheric CO2 [38,44], and industrial exhaust gasses pointed out that phosphate was not a limiting factor to biomass
[28] are often used as the carbon sources under photoautotrophic growth [17] due to the B. braunii's ability to accumulate large
conditions. However, the CO2 fraction in the air of about 0.03% [44] amounts of surplus phosphorus [68]. The source and concentra-
is not sufcient. Hence, an extra amount up to 2% (v/v) is needed tion of sulfur also inuence the growth of B. braunii. Concentra-
to obtain increased biomass (1.31.8 times) and palmitic and oleic tions of bisulte, sulte and sulfate up to 1 mmol/L, 3.2 mmol/L
acid (2.53 times) concentrations in B. braunii (LB 572) strain cells and 4 3.2 mmol/L, respectively showed no toxic effects on the
[65]. Of greater interest from the environmental and the biodiesel growth of B. braunii [32]. However, these experimental data did
production point of view is the use of industrial ue gas as a CO2 not indicate the inuence of sulfur source and concentration on
source, since B. braunii tolerates [66] the sultes and bisultes that the lipid content or productivity. Although not reported in Table 2,
are usually present in the ue gas. It is corroborated by the fact iron usually as FeCl3 at concentrations up to 0.74 mM - decreases
that at the ue gas rate of 0.3 v/v/m, the biomass and lipid (with biomass productivity under nitrogen-decient conditions [57].
an oleic acid fraction of 55%) productivity for B. braunii increases Additionally, it increases the lipid productivity (with highest
2.9-fold and 3.7-fold, respectively, compared to cultures cultivated fractions of palmitic and oleic acids) of other B. braunii strains
with ambient air containing 10% CO2 [28]. However, using ue gas (TRG, KB, SK and PSU). Other elements such as potassium, mag-
for the B. braunii cultivation in seawater impacts the marine eco- nesium and zinc are known to have an impact on algae cultivation
system, coastal area shing, boating and ship trafc. but have not been reported to be signicant for B. braunii biomass
Wastewaters are promising B. braunii cultivation media due to and lipid productivities. By comparing these nutrient values with
their enhanced nitrate and mineral contents. As can be seen in the reported ones (Table 2), we can predict the optimal medium
Table 2, wastewaters are rich in various nitrogen sources, such as for both the biomass and lipid (rich in palmitic and oleic acid
nitrate, ammonium, and nitrite. An earlier investigation showed content) productivities. For example, efuent from a secondary
that B. braunii rst consumes the nitrogen sources with the domestic and untreated seafood wastewater treatment systems
highest concentration in the medium [44]. However, when their are optimal for B. braunii (LEM14) and B. braunii (UTEX 572), re-
concentrations are equivalent, ammonium and nitrate uptakes are spectively. Wastewaters can also be sources of organic carbon. The
prioritized [67]. Furthermore, low nitrate levels in the nutrient cultivation of B. braunii in a molasses wastewater produced 1.3 and
medium (below 0.04 mM) lead to lower B. braunii biomass growth 1.2 times more biomass and lipids compared to photoautotrophic
rates, higher lipid content and lower lipid productivity of growth [44]. From the environmental point of view, the effect can
0.090 day  1, 63% and 0.009 (g/(L day)), respectively [25]. This be enhanced by adding both organic and inorganic carbon sources
lower biomass production has a negative effect at industrial levels, when cultivating B. braunii, which can solve the wastewater and
where lipid productivity is more important than lipid content [21]. industrial exhaust gasses problems. For instance, the CO2 supply in
High B. braunii (UTEX 572) biomass growth rates and lipid pro- a BG-11 medium enriched with glucose provides higher biomass
ductivities of 0.159 day  1 and 0.019 g/(L day), respectively can be and lipid yields [69].
achieved at a moderate nitrate concentration of 0.37 mM [25]. The optimal biomass and lipid productivities in synthetic and
However, a B. braunii (KMITL 2) strain showed a lower lipid pro- natural media can be obtained by controlling several para-
ductivity rate of 0.004 g/(L day) [64] with an almost equal nitrate meters: temperature, pH, light and mixing. Excepting two strains

Table 2
Common chemical composition and nutrient concentrations of various wastewaters (mg/L).

Ion Efuent from a secondary domestic Efuent from a secondary urban Mixa of carpet industry wastewaters Secondary seafood processing
wastewater treatment system wastewater treatment system and sewage plant wastewaterb

Untreated Treated

Na 28.98 112.1133.4 117.8151.5
NH4 12.76 15.00 17.5825.85 0.573.61 4.6
K 7.99 7.7510.32 9.7410.17
Mg2 2.63 13.8220.78 11.2813.21
Zn2 0.71 o0.05 0.030.13 0.170.18
NO3  0.2 0.90c 0.2128.13 1.393.91 24.1
PO43  2.00 11.50 20.3135.10 17.5921.95
Cl  1.70
SO42  4.21 86.96142-80 128.26182.10
References [39] [40] [43] [44]

Also contains small elemental phosphor and sulphur in both untreated and treated wastewater samples.
Also contains NO2  of 0.04 mg/L.
In total nitrogen content of 11.9 mg/L.
264 M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270

Table 3
Comparison between typical microalgae cultivation systems.

Open systems Closed systems Hybrid systems

Advantages 1. Relatively cheap 1. High biomass productivities 1. Minimal contamination due to controlled inoculum growth
2. Easy to clean 2. Suitable for outdoor cultures in the rst stage
3. Utilizes non-agricultural land 3. Good stirring 2. Sufcient seed supplies keep the preferred algal specie
4. Low energy inputs 4. Avoid water evaporation (s) dominant
5. Easy maintenance 5. Well controlled culture conditions
6. Easy to scale-up 6. Permits single strain culture
7. Increased the amount of light received
8. Lower level of contamination
Disadvantages 1. Low productivity 1. More expensive to construct and 1. Large-scale applications have been limited by the cost of the
2. Environmental factors are beyond operate rst stage
control 2. High energy inputs
3. Large area of land required 3. Requiring cooling
4. Limited to few strains of algae 4. Not easy scalable
5. Signicant evaporative losses 5. Labor intensive maintenance of bag
6. Poor Stirring, light and CO2 utilization systems
7. Cultures are easily contaminated 6. Not eliminates contamination
8. Limited recovery of biomass

(BOD-NG17 and BOD-GJ2) that show tolerance to extreme tem- harvested biomass, and lipid extraction. The downstream pur-
peratures of 40 C and  20 C [32], most B. braunii strains have an ication steps are: possible catalyst separation, alcohol recovery,
optimal growth temperature of 25 C and show satisfying re- nal product purication, and wastewater treatment. While the
sistance to temperature changes. This temperature tolerance can downstream purication steps are usually common for various
be substantial for the B. braunii outdoor mass cultivation because technologies, the upstream pretreatment phase is of crucial im-
problems like overheating and/or poor thermal (temperature) portance, and hence, it is further discussed.
control can be avoided. Furthermore, the temperature change
correlates strongly with CO2 solubility and indirectly with pH 5.1. Upstream pretreatment
maintenance. Higher temperatures decrease CO2 solubility and
increase pH, which favors the blooming of B. braunii [50,51]. On 5.1.1. Cultivation
the contrary, the reduction of pH results in a low biomass pro- B. braunii can be grown in aquatic environments or in non-
ductivity [44,65]. A wide pH range of 6.08.5 was tolerated only by agricultural land cultivation systems located near power plants
two B. braunii strains (LB-572 and SAG 30.81) [70] while the and/or wastewater treatment plants to access CO2 from ue gas
adaptation period of B. braunii (UTEX 572) was longer [28]. Ge and/or nutrient supplies, respectively. The goal of B. braunii culti-
et al. [71] reported that the B. braunii (765) strain performed os- vation is to mimic the natural environmental conditions which
moregulation by the synthesis/dissimilation of intracellular gly- favor algal growth and lipid productivity. Generally, according to
cerol, which maintained constant intracellular pH over a wide their design characteristics, B. braunii cultivation systems can be
range of the external pH. However, there is no data conrming classied as open or closed.
that lipid production by this algal strain is affected by the culture Open systems are outdoor facilities such as ponds (unstirred,
pH. For outdoor algae cultivation, besides temperature and pH, inclined, with a paddle wheel, and circulating ponds), lagoons,
solar radiation and light path have major impacts. The average deep channels, and shallow circulating units. Closed systems, also
recorded solar radiation during the outdoor cultivation of B. known as photobioreactors, include vessels, vertical-columns, os-
braunii is about 5 kW h/(m2 day) [21,37]. At constant solar radia- cillatory ow reactors, airlift reactors, xed or oating, helical
tion, higher biomass culture concentrations can be achieved if the (coils) or annular tubes, plates (panels or layers), and bags - all
light path is shorter [21]. On the other hand, if articial light is with walls made of transparent materials. Photobioreactors can
used with a 16:8 light:dark cycle, uorescent lamps are most ef- also be located outdoors, under sunlight or indoors, under articial
fective for increasing B. braunii (KMITL 2) lipid contents and yields irradiation facilities. In closed design systems, algae can also be
[64]. By contrast, blue-green LEDs lower costs for B. braunii (NIES- immobilized. Currently, there are six types of immobilization
836) cultivation [72]. However, improved pH, temperature dis- methods: covalent coupling, afnity immobilization, adsorption,
tribution and light intensity conditions are ineffective without connement in the liquid-liquid emulsion, capture behind semi-
appropriate mixing. For B. braunii strains, mixing is most fre- permeable membrane and entrapment. Five types of bioreactors
quently provided by CO2 aeration [69,71,73,74] or mechanically are used: uidized-bed, packed-bed, parallel-plate, air-lift, and
using a 15100 rpm velocity paddle wheel [24,38]. Compared to hollow-ber.
manually tested cultivations, the rapid (80100 rpm) paddle wheel Hybrid systems, which are a combination of open and closed
mixing provided higher lipid content of 48.4% [38] and lower oleic systems, are also reported in the literature [7577], but not for B.
acid content (19.2%). It leads to the conclusion that the highest braunii cultivation. Advantages and disadvantages of each culti-
biomass and lipid productivities were obtained under lower mix- vation system are summarized in Table 3.
ing intensity. Open systems. Four strains of B. braunii have been cultivated
in open systems for biodiesel production. B. braunii (AP103), B.
5. Biodiesel technologies braunii (LB 572) and two B. braunii strains isolated from Loktak and
Udaisagar lakes were cultivated in raceway pond [24], raceway and
Algae biodiesel technology includes three phases: upstream unstirred circular ponds [37] and plastic trays [37], respectively.
pretreatment, trans/esterication and downstream purication. Size capacities were: 0.710 m2 (2032000 L) and 1 m2 (287 L) for
The upstream pretreatment consists of the following steps: culti- the raceway and unstirred circular ponds, respectively. All these
vation in open and closed systems, harvesting, dewatering of experiments were photoautotrophic cultivations on synthetic
M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270 265

(modied Chu 13) medium with atmospheric CO2 as an inorganic high energy and total costs involved. The advantages and dis-
carbon source, thus making commercial applicability limited. advantages of each harvesting technique are well summarized in a
However, the cultivation time of 18 days was enough to produce recently published paper [83]. Since B. braunii grows in colonies,
approximately 1.85 g/L of B. braunii biomass, and mixing had the its harvesting is less challenging than for no-colony microalgae
main inuence on the lipid product quantity and quality. The [84]. So far, laboratory scale occulation [26,85] and in situ mag-
optimal mixing rate was 15 rpm [24], which induced 2024% of netic [31] harvesting have been tested for B. braunii cells rich in
lipid content with 34% of oleic acid. Mosquitos can severely affect lipids. B. braunii (UTEX 572) cells had been harvested using various
B. braunii outdoor biomass production if the nutrient medium is occulation methods. The pH adjustment and addition of various
not supplemented with 0.5 M NaCl [63]. salts such as Al2(SO4)3, NH4Cl, KCl, CaCl2, MgSO4, and FeCl3 [26,85]
Currently, open systems are not implemented on commercial or bioocculants such as Pestan and that produced by the bac-
scales for the B. braunii cultivation intended for biodiesel pro- terium P. polymyxa AM49 [26,85] are just a few noteworthy. The
duction, due to numerous unanswered questions. For example: most efcient method for recovery (with the occulating activity
(i) Can we improve the low productivity of open systems by im- of 90.6%) was treatment with both FeCl3 and the bioocculant
plementing nutrient-rich wastewaters, using large inoculum or produced by the bacterium P. polymyxa AM49. The same recovery
frequent feeding/harvesting. (ii) Does the covering of open ponds efciency of 90% was achieved with an in situ magnetic separation
with permeable plastic covers or greenhouses provide better system [31]. Although the former method has relatively low cost
control over water evaporation, atmospheric temperature, wind and no cells damage, it has the following major challenges for
speed, net solar radiation as well as transfer and supply of CO2? commercial applications: high energy inputs, the possibility of
(iii) What is the quality of water obtained after algal biomass de- bioocculants agents competing for medium nutrients, and the
watering? Is it compatible with the optimal growth composition? inability to recycle water back into the culture system. On the
(iv) Is the Submersible aquatic algae cultivation technology [67] other hand, in situ magnetic harvesting suffers from high pro-
adaptable for the B. braunii strains? duction costs associated with the high cost of CO2 consumption
and capital investments. Therefore, different harvesting techni- Closed systems. Photobioreactors implemented in B. braunii ques must be integrated to increase the efciency of microalgae
production use both suspended and immobilized microalgae cells. harvesting. For example, electro-occulation and dispersed air
Suspended B. braunii (LB572) and B. braunii (IPE 001) cells are otation were integrated for harvesting B. braunii [86] to improve
cultivated in the panel [21] and plastic bag [31] reactors, respec- the biomass recovery signicantly. The recovery efciency of 98.9%
tively. Also, the cultivation of suspended B. braunii (NIES 2199) was reached within only 14 min. Future investigations should
cells in a submerged membrane photobioreactor using efuent determine the energy and cost demands of this method as well as
off-gas (CO2) from an aeration tank and nutrients from the treated the possibility for its scaling-up.
sewage was modeled [78]. Novel immobilized cultivation systems
with thick concrete layers and wood support plates [36] or biolm 5.1.3. Dewatering of harvested biomass
layers [29,30] are also used for cultivating B. braunii (LB 572), B. Some processes, such as in-situ transesterication or hydro-
braunii (FACHB 357) and B. braunii (SAG 807-1). Capacities of 400 thermal liquefaction (HTL), need no or only partial dewatering of
1000 L and 200 L for the panel and plastic bag reactors, respec- harvested biomass. In all other cases, extensive biomass dewater-
tively, have been used only for suspended cells while immobilized ing is required due to the possible inhibition of subsequent pro-
cells are still at the laboratory scale. Like open systems, all of these cesses such as lipid extraction and trans/esterication. Also, de-
experiments were conducted using synthetic media with atmo- watering temperature strongly affects both lipid composition and
spheric CO2 as an inorganic carbon source [31] and using fertilizers yield. Commonly used methods to dry the wet biomass paste are:
[21]. Immobilized biolm cultivation gives much lower biomass solar, low-pressure shelf, spray, drum, uidized-bed, freeze, and
productivity compared to open pond systems, but the highest one Refractance Window drying [80]. However, dryers are high
(5.5 g/(L day)) among closed systems. However, the maximum li- energy-consuming devices and, therefore, this type of dewatering
pid productivity of 20.36 g/(L day) was obtained with a plastic bag of harvested biomass can seriously jeopardize the commercial
photobioreactor under outdoor ultrasonic-treated conditions. All viability of microalgae biodiesel production. Little information
of these studies indicated that linoleic and oleic acids are major about the development of efcient dryers for the B. braunii bio-
lipid components. mass is available in the literature.
Although the use of non-agricultural land for closed systems is
far less than for open systems, current photobioreactor technolo- 5.1.4. Lipid extraction
gies have not yet been implemented for cultivating B. braunii on For feasible extraction of lipids, a good starting point is to
commercial scales. For example, outdoor plastic bag cultivation measure total lipids from B. braunii using Nile Red (9-diethyla-
faces problems with high operating costs, limited recovery of mino-5H-benzo[]phenoxazine-5-one) staining [87]. After this
biomass, and light availability. On the other hand, limitations of inexpensive and uncomplicated method, lipid recovery can be
immobilized biolm cultivation include the high density of im- carried out in two steps that include cell disruption and lipid ex-
mobilized matrix reducing the light penetration, its high cost and traction. There are numerous disruption methods, which are di-
limited scalability beyond the laboratory. A very interesting closed vided into four categories: mechanical, physical, chemical, and
system design for cultivating green microalgae (Desmodesmus sp.) enzymatic. Each category is widely explained and well summar-
using wastewaters is the Offshore Membrane Enclosures for ized elsewhere [88]. Since lipids of B. braunii are accumulated in
Growing Algae (OMEGA) system [79]; however, there has been no vacuoles, they can easily be released by slight cell disruption. At
report on its application for B. braunii cultivation. the laboratory scale, B. braunii cells are mainly disrupted me-
chanically [60]. It seems that this disruption method does not af-
5.1.2. Harvesting fect the extracted lipid yield since the same yield is obtained with
Microalgae harvesting is a two-stage process involving: (i) bulk lysed and whole cells. The effect of this disruption method on the
harvesting, such as occulation, otation, or gravity sedimenta- lipid composition has not yet been investigated.
tion; and (ii) thickening, such as centrifugation, ltration, or ul- Lipid extraction methods, such as solvent extraction, SC uid
trasonic aggregation [8082]. Harvesting is a challenging phase extraction, HTL, and milking in microalgal biodiesel production are
due to the low densities and small sizes of algal cells, as well as the worthy of mention [60,8992]. The advantages and disadvantages
266 M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270

of different methods for lipid extraction have been widely studied possible improvements are yet to be studied.
[93,94]. Amongst all, SC uid extraction is the most efcient for B.
braunii lipid extraction. Similar FAME yields per ash-free dry 5.3. Downstream purication
weight (AFDW) of 70.85 mg/g AFDW and 60 mg/g AFDW [60] were
obtained from lipids extracted with hexane and SC CO2 extraction For commercial acceptance, biodiesel produced from micro-
methods, respectively. Hence, the latter was shown to be more algae needs to meet currently existing fossil fuel standards. It
selective for the extraction of neutral lipids [60,95], which are means that biodiesel needs to be separated from the trans/ester-
necessary for biodiesel production. Also, the harvested B. braunii ication reaction mixture and further puried to reach the stan-
cells can be thermochemically liqueed, yielding an oil (64%), dard quality. Technologies such as gravitational settling, micro-
aqueous, gaseous (CO2), and tar fractions [91]. However, in this wave irradiation, centrifugation, membrane ltration, wet/dry
research, the oil was not characterized, so it is questionable if it washing, and membrane rening are often included in biodiesel
can be applied to biodiesel production. It is interesting to mention separation and purication [103106]. However, separation and
the milking of B. braunii Ktz (CCAP 807/2) cells in both solvent- purication studies of B. braunii biodiesel have not been revealed
based and the solvent-free (blotting) forms, as they are non-de- so far. Furthermore, the biorenery concept must use a zero-waste
structive external oil extraction methods which cause no cell da- process, so the utilization of by-product process streams is crucial
mage [90]. It means that after lipid extraction, the cells can be for plant facility design. If the process ows - for example, lipid
returned to the cultivation medium to continue synthesizing li- extracted microalgal biomass residues - can not be recycled into
pids. However, this method is not signicant for biodiesel pro- the process, they should be converted into biofuels like bio-
duction due to low TAG yields (o6.65 mg/g AFDW and methane, bioethanol, biohydrogen, biobutanol [107] or other va-
o0.08 mg/g AFDW for the 20 min heptane and the 3 h blotting luable products like drugs, food, feed additives [108], and animal
methods, respectively). However, since milking efciently extracts feed [109]. The use of combustion, gasication, pyrolysis, lique-
hydrocarbons (495%) it is of enormous importance for obtaining faction, carbonization, and anaerobic digestion of the algal bio-
renewable, white and Hydro-Thermal-Upgraded (HTU) diesels. mass residue are widely cited in the literature. Nevertheless, the
Currently, SC uid extraction is only conducted at pilot scale lipid-extracted residue biomass of B. braunii (BOT-22) has been
because of limitations caused by high energy and economic costs. investigated only as a feedstock for steam pyrolysis producing a
However, scale-up may not be a necessary step, since quantities hydrogen-rich solid fuel which is comparable to fossil fuel [110].
produced in short timeframes from cultivators are in kilogram-
scale. Lastly, it remains to be seen how efcient one-step dewa-
tering, cell disruption, and lipid extraction techniques, such as the 6. The quality of biodiesel obtained from B. braunii's lipids
Origin Oil single step extraction method [92], will be for B. braunii
lipid extraction in biodiesel production. Although this alga is mainly studied for hydrocarbon produc-
tion, recently published papers conrm its application for biodie-
5.2. Trans/esterication sel production, too. A signicant number of biodiesel physico-
chemical properties were observed to be similar to that of petro-
After the extraction of lipids from microalgae, the trans/ester- leum diesel fuel. For instance, B. braunii (UTEX and IBL-C117)
ication process is necessary to produce high-quality biodiesel. showed better matching with all three International standards
Trans/esterication is the most studied biodiesel production phase (EN14214, ASTM D6751-10, RANP/2008) values regarding CN, cold
of the last decade. Comprehensive pathways of microalgae bio- lter plugging point (CFPP), and (IV) [111]. Even better fuel prop-
mass trans/esterication have already been investigated erties results were found in B. braunii (KMITL) strain due to lower
[73,81,83,9698]. Methanol and ethanol are frequently used, IV and degree of unsaturation (DU) values which are signicant for
especially the former one, for producing biodiesel and glycerol. oxidative stability of biodiesel [112]. However, all three B. braunii
The trans/esterication process may occur in the presence of a strains were cultivated in synthetic media which inuence the FAs
base, acid, enzyme, or with no catalyst. The optimal reaction time proling and consequently the quality of biodiesel. Hence, a major
to achieve a high lipid conversion depends on the operating con- boost in the technology of B. braunii biodiesel synthesis will be
ditions and the catalyst type and varies from one minute [99] to producing high-quality fuel from natural media which will meet
two days [100]. Due to the incomplete lipid extraction from the B. International standard directives.
braunii biomass (high contents of residual biomass rich in lipids),
improvements to existing methods are directed towards the in-
tegrated concept of simultaneous extraction and trans/esterica- 7. Economic assessment and life cycle analysis
tion, also known as direct trans/esterication or in-situ trans/es-
terication (ISTE) [89,101]. ISTE in batch and reux extraction re- The biggest obstacle for the commercial production of biodiesel
actors in semi-continuous laboratory scales are performed under from algal biomass is its high price (US$ 2.76/kg), which is nearly
solvent/cosolvent/catalyst [89,101] conditions. The highest FAME triple that of fossil diesel (US$ 0.95/kg) [113]. This price is due to
yield of 480% is obtained in a reux extraction reactor using the complex, hardly scalable and costly processing steps, such as cul-
homogenous H2SO4 catalyst and a low solvent/cosolvent ratio (3:1 tivation, harvesting, dewatering of biomass, and lipid extraction.
methanol/chloroform). ISTE is a single-step process which reduces Table 4 compares the cost of various production phases using B.
both capital and energy costs. Moreover, it is most effective for braunii and other microalgal species.
measuring total fatty acids in microalgae [89], which are essential This table indicates that the costs associated with open ponds
for biodiesel production. However, the high biomass water content are the lowest. Furthermore, the crucial economic problem to be
signicantly decreases the biodiesel yield, and the presence of solved is to develop low-cost harvesting and lipid extraction
cosolvents/catalysts makes downstream processing more complex. methods. It is because harvesting, drying and extracting techni-
Although it is not employed in the case of the B. braunii, ISTE under ques represent about half of the total production cost [97]. Har-
SC conditions [102] is a good alternative to classic ISTE. The SC ISTE vesting alone amounts to 30% of the total cost of biodiesel pro-
method is a one-step process for direct liquefaction and conver- duction from microalgae [14] and can be as high as about US$ 10/
sion of wet algal biomass without using cosolvent/catalyst mix- kg [73]. To be competitive with fossil diesel, the cost of extracted
tures. However, the techno-economic feasibility of SC ISTE and its algal lipids should be:
M.B. Tasi et al. / Renewable and Sustainable Energy Reviews 64 (2016) 260270 267

Table 4 each nutrient on lipid composition (especially FAs and TAGs) is a

Costs of the production phases for B. braunii and some other algae. major disadvantage. Also, a systematic approach to optimizing the
medium nutrition composition for the selected producing strain is
Costs References
necessary for its commercial utilization. It means that much ex-
Cultivation B. braunii (IRE 001) US$ 20.76/kga [31] perimentation is required to determine the inuence of each nu-
Reports on other taxonomic US$ 1.98/L to US$ [114] trient type and concentration on the biomass and lipid pro-
groups of algae 5.50/Lb,c ductivities, as well as on the lipid composition of each growth
US$ 3.30/L to US$
phase for the specied strain of B. braunii. This information will
US$ 0.60/kg and US$ [115,116] help in concluding if a natural medium, such as wastewaters, is
1.54/kgc useful as the cultivation medium for B. braunii strains which will
US$ 0.47/kg and US synthesize lipids for high-quality biodiesel.
$ 7.32/kga
The design of cultivation systems has also been widely studied.
US$ 1.02/kgd [116]
The open pond is proven to be viable for industrial cultivation of B.
braunii. The problems associated with the open pond cultivation,
Harvesting B. braunii (IRE 001) US$ 2.07/kge [31]
such as contamination or unsustainable supply of fertilizer, can be
Reports on other taxonomic US$ 0.07fUS$ 0.22/ [117]
groups of algae Lg minimized. It is because B. braunii: (i) grows in colonies; (ii)
US$ 0.00011/Lh [86] blooms, suggesting allelopathic effects; and (iii) can be easily
grown in wastewaters rich in nutrients with a supply of CO2. Also,
Extraction Reports on other taxonomic US$ 0.02US$i 0.06/L [117] medium densities of Chlorella vulgaris and Chlamydomonas re-
groups of algae inhardtii stimulate the growth of B. braunii. However, the con-
US$ 1.87/L [118] siderable amount of land for open ponds, the low biomass yields
US$ 2.80/L [115] and the evaporation should be addressed in the future works.
US$ 3.98/L [118]
Closed loop systems do not have the problems of open-pond
Photobioreactors. systems, but they cannot easily be scaled, are not cheap to con-
Biodiesel price. struct and consume more energy. New methods for B. braunii
Open ponds. biomass harvesting and lipid extraction should further be in-
e vestigated due to their signicant shares of the total production
In situ magnetic harvesting.
Algae Venture Systems's harvesting technology. costs. Reducing the number of steps by introducing integrated
Centrifuge. phases, such as SC ISTE, is essential to provide easier, better and
Electroocculation. lower cost systems. Also, while laboratory experiments have
Algae to Energy Inc. patented system. mostly been reported for harvesting and trans/esterication, these
production phases are yet to be tested further at the pilot and
Calgal lipid = 8.48104 Cfossil diesel (1) industrial scales. The biodiesel must be thoroughly examined re-
garding its quality as a fuel, and no hazardous wastes should be
where, C algal lipid is the price of algal lipid (US$/L) and C fossil diesel is released during downstream processing. It means that more work
the price of crude fossil diesel (US$/L), assuming that algal lipids on the economic feasibility and LCA of biodiesel production from
have 80% of the caloric value of crude fossil diesel [115]. It means B. braunii should be done.
that a reasonable algal lipid price should be US$ 0.48/L. To the best
authors knowledge, the costs of B. braunii extracted lipids have
not been reported yet. Acknowledgments
Besides economic benets, the ecological footprint obtained by
life cycle analysis (LCA) is another advantage and conformation of This study was supported by Fundao de Amparo Pesquisa
the environmental sustainability of microalgae-based biodiesel. do Estado de So Paulo (Process No. 14/18979-6) and the Ministry
Among a few LCA reports considering algal species [119123], of Education, Science and Technological Development of the Re-
there is one dealing with a report on the LCA for B. braunii used in public of Serbia (Project III 45001). The authors are particularly
hydrocarbon production [124]. thankful to Dan S. Taranu, Ph.D. and Marija D. Stankovi, Ph.D. for
revision of the English and additional comments.

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