2. Physiology and biochemistry of Botryococcus braunii

B. braunii races differ also by its morphological and physiological characteristics. Cells from A and B races are of 13 μm 7–9 μm, and those of L race are 8–9 μm 5 μm [19].

Each colony is constituted by a group of 50–100 piriform cells embedded in a hydrocarbon network and the extracellular matrix (ECM). This ECM contains three main components: (1) a fibrous cell wall surrounding each cell and having β-1,4 and/or β-1,3-glucans including cellulose; (2) the intracolonial space constituted by a network of liquid hydrocarbons; and (3) a fibrillary sheath composed mainly of arabinose and galactose polysaccharides, holding the liquid hydrocarbons [20].

B. braunii may have a hetero-, mixo-, or phototrophic grow and the morphology will depend on the C source and the amount of light [21]. The hydrocarbon production is associated with the cell division [22], likely due to the localization of the enzymes involved in the alkadienes, alkatrienes (race A), and botryococcenes (race B) biosynthesis [23].

Other difference among the races is the keto-carotenoid accumulation in the stationary phase of cultures. Races B and L change color from green-brown to orange, and race A changes from green to yellow-orange [1]. The production of carotenoids is also a stress response by environmental factors. The DAD1 gene expression, a suppressor of programmed cell death, was reported in race B, under stress conditions at 10–60 min [24]. B. braunii is tolerant to desiccation and extreme temperatures, which allows its global dispersion in different environments [25]. The reproduction mechanism of B. braunii seems to be autosporic [26].

Symbiotic bacteria have been reported after microscopic observations, and an ectosymbiont α-proteobacteria (BOTRYCO-2) that promotes the productivity of biomass and hydrocarbons was described [2, 27].

### 2.1 Biosynthesis of alkadienes and alkatrienes

Characteristic alkadienes and alkatrienes of race A have double links and similar stereochemistry as oleic acid. Experiments with labeled fatty acids have shown that this one is the main precursor by the long-chain fatty acids (LCFAs) pathway, followed by a decarboxylation process [1, 17, 28, 29]. The first step is the elongation of oleic acid (18:1 cis-Δ9) and its isomer elaidic acid (18:1 trans-Δ9). The acyl-CoA reductase and decarbonylase enzymes in race A microsomes suggest an alternative mechanism where the LCFAs are reduced to aldehydes and decarbonylated to produce alkadienes and alkatrienes [17, 30]. Race A transcriptome allowed the identification of six candidate genes potentially involved in this biosynthesis [31].

#### 2.2 Biosynthesis of botryococcenes

The analysis of race B transcriptome and other evidences suggests that the biosynthesis of isoprenoids comes from the deoxyxylulose phosphate/methylerythritol phosphate (DXP/MEP) pathway [32–34]. Expressed sequence tag (EST) markers for enzymes of the DXP/MEP pathway [34], as well as multiple isoforms of

Addition of another IPP forms the geranylgeranyl diphosphate (GGPP), precursor of the tetraterpenoid carotenoids (Figure 3b). This begins with the formation of a trans-isoprenyl diphosphate by the phytoene synthase (CtrB) enzyme, condensing two GGPP molecules in two steps with the release of pyrophosphate. In the first step, (1R, 2R, 3R)-prephytoene diphosphate is produced from half

converted into a wide variety of carotenoids [34, 36–38]. All are important antioxidant photoprotectors and modulators of the function of membrane proteins for

The squalene production [40] starts with the Botryococcus squalene synthase (BSS) enzyme, using two FPP molecules. Botryococcenes production uses also two FPP molecules but the product is the intermediary cyclopropyl presqualene diphosphate (PSPP) (Figure 3c). With NADPH, the PSPP has two options; one forms the botryococcene with a C3-C1 connection between the FPP molecules (Figure 3d). The other option forms a C1-C1<sup>0</sup> between two FPP molecules producing squalene

Lycopadiene biosynthetic pathway. (a) Reduction of GGPP to PPP and condensation by LOS. (b) LOS condensation of GGPP to form phytyl diphosphate and reduction to lycopaoctaene. (c) FPP use by LSS or LOS for squalene production. DXR, 1-deoxy-D-xylulose-5-phosphate reductase; DXS, 1-deoxy-D-xylulose-5 phosphate synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GPPS, geranyl diphosphate synthase; GPPR, geranyl diphosphate reductase; NADPH<sup>+</sup> and

, nicotinamide adenine dinucleotide phosphate (reduced and oxidized); PPi, inorganic pyrophosphate; PPP, phytyl diphosphate; PLPP, prelycopaoctaene diphosphate; LOS, lycopaoctaene synthase; LSS, B. braunii


cyclopropyl (C10

Figure 4.

NADP<sup>+</sup>

127

race L squalene synthase. Adapted from [15].

photosynthetic complexes [39].

The Colonial Microalgae Botryococcus braunii as Biorefinery

DOI: http://dx.doi.org/10.5772/intechopen.88206

#### Figure 3.

Biosynthesis of tri- and tetraterpenes in B. braunii race B. (a) FPP production; (b) carotenoid production from GGPP; (c) squalene production from FPP; (d) methylated botryococcene production; (e) methylated squalene production. BSS, Botryococcus squalene synthase; CtrB, phytoene-synthase; DXR, 1-deoxy-D-xylulose-5 phosphate reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FPPS, farnesyl diphosphate synthase; GPPS, geranyl diphosphate synthase; NADPH<sup>+</sup> and NADP<sup>+</sup> , nicotinamide adenine dinucleotide phosphate (reduced and oxidized); PPi, inorganic pyrophosphate; PSPP, cyclopropyl presqualene diphosphate; SAM, S-adenosyl methionine; SAH, S-adenosyl-L-homocysteine; SSL, squalene synthase-like; SMT, squalene methyltransferase; TMT, triterpene methyltransferase. Adapted from [17, 34].

enzymes for the 3-phospho-D-glycerate biosynthesis from D-glyceraldehyde-3 phosphate and pyruvate as precursors, were identified. Some of the respective transcripts are present in high abundance (>250 reads/Kb), suggesting a high metabolic flow in B. braunii [31].

The first step is the formation of 1-deoxy-D-xylulose-5-phosphate (DOXP) by the DOXP synthase (DXS) (Figure 3).

The characterization of three DXS isoenzymes in race B shows that they are active and have similar kinetic parameters, which increases the metabolic flow for the production of terpenoids [35]. The DOXP is reduced by the DXP reductoisomerase (DXR) to 2-C-methylerythritol-4-phosphate (MEP), and converted to isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In the B. braunii transcriptome, only one DXR has been found [34]. The next step involves condensation of IPP and DMAPP to form geranyl diphosphate (GPP), and the addition of other IPP produces farnesyl diphosphate (FPP) [17] (Figure 3a). Two B. braunii genes code for farnesyl diphosphate synthase isoenzymes (FPPS) with an amino acid identity of 72% [34].

The Colonial Microalgae Botryococcus braunii as Biorefinery DOI: http://dx.doi.org/10.5772/intechopen.88206

Addition of another IPP forms the geranylgeranyl diphosphate (GGPP), precursor of the tetraterpenoid carotenoids (Figure 3b). This begins with the formation of a trans-isoprenyl diphosphate by the phytoene synthase (CtrB) enzyme, condensing two GGPP molecules in two steps with the release of pyrophosphate. In the first step, (1R, 2R, 3R)-prephytoene diphosphate is produced from half cyclopropyl (C10 -2-3) reordered to provide 15-cis-phytoene, which can be converted into a wide variety of carotenoids [34, 36–38]. All are important antioxidant photoprotectors and modulators of the function of membrane proteins for photosynthetic complexes [39].

The squalene production [40] starts with the Botryococcus squalene synthase (BSS) enzyme, using two FPP molecules. Botryococcenes production uses also two FPP molecules but the product is the intermediary cyclopropyl presqualene diphosphate (PSPP) (Figure 3c). With NADPH, the PSPP has two options; one forms the botryococcene with a C3-C1 connection between the FPP molecules (Figure 3d). The other option forms a C1-C1<sup>0</sup> between two FPP molecules producing squalene

#### Figure 4.

enzymes for the 3-phospho-D-glycerate biosynthesis from D-glyceraldehyde-3 phosphate and pyruvate as precursors, were identified. Some of the respective transcripts are present in high abundance (>250 reads/Kb), suggesting a high met-

(reduced and oxidized); PPi, inorganic pyrophosphate; PSPP, cyclopropyl presqualene diphosphate; SAM, S-adenosyl methionine; SAH, S-adenosyl-L-homocysteine; SSL, squalene synthase-like; SMT, squalene

Biosynthesis of tri- and tetraterpenes in B. braunii race B. (a) FPP production; (b) carotenoid production from GGPP; (c) squalene production from FPP; (d) methylated botryococcene production; (e) methylated squalene production. BSS, Botryococcus squalene synthase; CtrB, phytoene-synthase; DXR, 1-deoxy-D-xylulose-5 phosphate reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FPPS, farnesyl diphosphate synthase;

, nicotinamide adenine dinucleotide phosphate

The first step is the formation of 1-deoxy-D-xylulose-5-phosphate (DOXP) by

The characterization of three DXS isoenzymes in race B shows that they are active and have similar kinetic parameters, which increases the metabolic flow for the production of terpenoids [35]. The DOXP is reduced by the DXP reductoisomerase (DXR) to 2-C-methylerythritol-4-phosphate (MEP), and converted to isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In the B. braunii transcriptome, only one DXR has been found [34]. The next step involves condensation of IPP and DMAPP to form geranyl diphosphate (GPP), and the addition of other IPP produces farnesyl diphosphate (FPP) [17] (Figure 3a). Two B. braunii genes code for farnesyl diphosphate synthase isoenzymes (FPPS) with an

abolic flow in B. braunii [31].

Figure 3.

amino acid identity of 72% [34].

126

the DOXP synthase (DXS) (Figure 3).

GPPS, geranyl diphosphate synthase; NADPH<sup>+</sup> and NADP<sup>+</sup>

Microalgae - From Physiology to Application

methyltransferase; TMT, triterpene methyltransferase. Adapted from [17, 34].

Lycopadiene biosynthetic pathway. (a) Reduction of GGPP to PPP and condensation by LOS. (b) LOS condensation of GGPP to form phytyl diphosphate and reduction to lycopaoctaene. (c) FPP use by LSS or LOS for squalene production. DXR, 1-deoxy-D-xylulose-5-phosphate reductase; DXS, 1-deoxy-D-xylulose-5 phosphate synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GPPS, geranyl diphosphate synthase; GPPR, geranyl diphosphate reductase; NADPH<sup>+</sup> and NADP<sup>+</sup> , nicotinamide adenine dinucleotide phosphate (reduced and oxidized); PPi, inorganic pyrophosphate; PPP, phytyl diphosphate; PLPP, prelycopaoctaene diphosphate; LOS, lycopaoctaene synthase; LSS, B. braunii race L squalene synthase. Adapted from [15].

that will be methylated (Figure 3e) further on. These reactions are catalyzed by squalene synthase-like (SSL) enzymes. Three SSL genes have been identified but none is directly related with the botryococcene biosynthesis [41]. However, when the 3 SSLs enzymes were mixed in vivo and in vitro, botryococcene (SSL-1 + SSL-3) or squalene (SSL1 + SSL-2) was synthesized. SSL-1 condenses two FPP molecules to produce PSPP [42], demonstrating the versatility and potential for metabolic engineering of botryococcene biosynthesis.

engines and cause corrosion, erosion, and accumulation of deposits in the nozzles; because of these reasons, they are mixed with standard fuels [49, 50]. B. braunii accumulates hydrocarbons similar to those of the crude oil, and their direct contribution in the formation of oil reserves currently in use has been reported [3–5]. The B. braunii oils showed almost equal values in density and surface tension than the diesel, but with higher kinematic viscosity and distillation temperature [50]. The B. braunii race B oil was already converted into diesel with an 85% performance, using a simple conversion process under mild conditions of 260°C and 1 atm. The physical properties are relatively close to the specification for diesel, with 40 as estimated

The Colonial Microalgae Botryococcus braunii as Biorefinery

DOI: http://dx.doi.org/10.5772/intechopen.88206

The limitation to use B. braunii as biorefinery is the slow growth rate of days in comparison with hours in other algae [49, 52]. Other factors affecting the growth

St Culture conditions SCGR Dt THC Ref.

Showa (B) 30 850 14:10 1 0.5 1.40 NIA [54] Showa (B) 25, 30 85–398 14:10 1.0–10.0 0.19–0.44 1.60–3.60 30–39 [54] Showa (B) 23–25 250 24 0.3 0.42 1.70 24–29 [52] Showa (B) 23 150 16:8 2 0.17 4.08d 25 [55] Yayoi (B) 25 240 12:12 2 0.2 3.50 40.5 [38] AC759 (B) 23 150 16:8 2 0.07 9.90<sup>d</sup> 21 [55] AC761 (B) 23 150 16:8 2 0.11 6.30<sup>d</sup> 45 [55] IPE001 (B) 25 35 16:8 1 0.15c 4.50<sup>c</sup> 64.3 [61] BOT-144 (B) 25 60<sup>a</sup> 24 0 0.16 4.33<sup>d</sup> 50 [62] LB-572 (A) 26 12 Klux 24 2 0.07<sup>c</sup> 10.60<sup>c</sup> 28 [53] Gottingen 807/1 (A) 25 26b 14:10 1 0.3 2.30 40.5 [67] AC755 (A) 23 150 16:8 2 0.05 13.86<sup>d</sup> 16 [55] CCALA777 (A) 23 150 16:8 2 0.06 11.55<sup>d</sup> 10 [55] CCALA778 (A) 23 150 16:8 2 0.17 4.08d 0 [55] CCAp807/2 (A) 23 150 16:8 2 0.11 6.30<sup>d</sup> 7 [55] 765 25 150 24 20 0.13c 5.50<sup>c</sup> 24 [64] 765 25 120 24 ASLW NIA NIA 23.8 [65] GUBIOTJTBB1 25 35 16:8 0 0.112 6.19 52.6 [66] AP 103 23 30 16:8 0 NIA NIA 13 [67] ASLW, aerated swine lagoon wastewaters (not sterile); °C, temperature; CO2, % v/v; Dt, doubling time (days); NIA, no information available; PAR, photosynthetic active radiation (μmols of photons/m<sup>2</sup> s); Php, photoperiod (light/ dark hours); SCGR, specific cell growth rate (μ/day); μ, specific velocity of growth rate; St, strain (race); THC, total

Comparison of culture conditions and productivity of hydrocarbons between B. braunii strains at laboratory

°C PAR Php CO2

cetane (CN) number [51].

hydrocarbons (% DW, dry weight).

Calculated values from μ, using Ln(2)/μ equation.

Blue light <sup>λ</sup> = 470 nm. <sup>b</sup>

Estimated values [54].

a

d

W/m<sup>2</sup> . <sup>c</sup>

Table 1.

scale.

129

Most botryococcenes are excreted to the ECM where they are methylated. The di- and tetramethyl forms are related to six genes coding for triterpene and squalene methyltransferases (TMT, SMT) [43] (Figures 3d and 3e). The botryococcenes are methylated to produce C31–C37 hydrocarbons, C34 being the main in race B. Three cyclic botryococcene C33 molecules and a trimethylsqualene isomer were recently found [44]. Also, two squalene epoxidase (BbSQE-I and -II) enzymes converting squalene into membrane sterols were identified [45]. Data of the B. braunii race B nuclear genome will allow the search for possible regulatory routes of this singular metabolism [46].

#### 2.3 Biosynthesis of lycopadiene

The formation of lycopadiene of race L is similar to the squalene. In the B. braunii transcriptome, there are two homologous contigs to squalene synthase (SS) [31]. One encodes a squalene synthase (LSS) and the other for a lycopaoctaene synthase (LOS). LOS uses preferentially in vivo GGPP, and C15 and C20 prenyl diphosphates as substrates [15] (Figure 4).

There are two biosynthetic mechanisms for lycopadiene from C20 prenyl diphosphate intermediates. In one, the GGPP reduction by a GGPP-reductase produces phytyl diphosphate (PPP), and LOS condenses two PPP molecules producing lycopadiene (Figure 4a). In the other one, LOS condenses two GGPP molecules producing prelycopaoctaene diphosphate (PLPP), which rearranges into lycopaoctaene. Finally, lycopadiene seems to be produced by enzymatic reductions not yet identified (Figure 4b).

LOS may also form squalene from FPP (Figure 4c). These results show the plasticity of L race to synthesize squalene and lycopadiene.

#### 2.4 Extracellular matrix (ECM) polymers

ECM contains long chains of polymerized polyacetal hydrocarbons joined to specific hydrocarbons of each race. There is a fibrillary sheath that envelops the entire colony, formed mainly by arabinose (42%) and galactose (39%). The cell wall contains β-1,4 and/or β-1,3 glucans making a cellulose-like polymer [20].

Also, there's a biopolymer resistant to nonoxidative chemical degradation as acetolysis. This biopolymer resembles sporopollenins [1] of the outer walls of pollen grains and spores of microorganisms [47]. It seems to be formed by oxidized carotenoid polymers and phenolic compounds that absorb UV-B light as p-coumaric and p-ferulic acids [48].

### 3. Profitability of B. braunii derivatives

#### 3.1 Hydrocarbons

Both bioethanol and biodiesel have a poor oxidative stability, low energy content by volume, and high content of oxygenated compounds, which damage combustion

### The Colonial Microalgae Botryococcus braunii as Biorefinery DOI: http://dx.doi.org/10.5772/intechopen.88206

that will be methylated (Figure 3e) further on. These reactions are catalyzed by squalene synthase-like (SSL) enzymes. Three SSL genes have been identified but none is directly related with the botryococcene biosynthesis [41]. However, when the 3 SSLs enzymes were mixed in vivo and in vitro, botryococcene (SSL-1 + SSL-3) or squalene (SSL1 + SSL-2) was synthesized. SSL-1 condenses two FPP molecules to produce PSPP [42], demonstrating the versatility and potential for metabolic engi-

Most botryococcenes are excreted to the ECM where they are methylated. The di- and tetramethyl forms are related to six genes coding for triterpene and squalene methyltransferases (TMT, SMT) [43] (Figures 3d and 3e). The botryococcenes are methylated to produce C31–C37 hydrocarbons, C34 being the main in race B. Three cyclic botryococcene C33 molecules and a trimethylsqualene isomer were recently found [44]. Also, two squalene epoxidase (BbSQE-I and -II) enzymes converting squalene into membrane sterols were identified [45]. Data of the B. braunii race B nuclear genome will allow the search for possible regulatory routes of this singular

The formation of lycopadiene of race L is similar to the squalene. In the B. braunii transcriptome, there are two homologous contigs to squalene synthase (SS) [31]. One encodes a squalene synthase (LSS) and the other for a lycopaoctaene synthase (LOS). LOS uses preferentially in vivo GGPP, and C15 and C20 prenyl

There are two biosynthetic mechanisms for lycopadiene from C20 prenyl diphosphate intermediates. In one, the GGPP reduction by a GGPP-reductase produces phytyl diphosphate (PPP), and LOS condenses two PPP molecules producing lycopadiene (Figure 4a). In the other one, LOS condenses two GGPP molecules

lycopaoctaene. Finally, lycopadiene seems to be produced by enzymatic reductions

LOS may also form squalene from FPP (Figure 4c). These results show the

ECM contains long chains of polymerized polyacetal hydrocarbons joined to specific hydrocarbons of each race. There is a fibrillary sheath that envelops the entire colony, formed mainly by arabinose (42%) and galactose (39%). The cell wall

Also, there's a biopolymer resistant to nonoxidative chemical degradation as acetolysis. This biopolymer resembles sporopollenins [1] of the outer walls of pollen grains and spores of microorganisms [47]. It seems to be formed by oxidized carotenoid polymers and phenolic compounds that absorb UV-B light as p-coumaric

Both bioethanol and biodiesel have a poor oxidative stability, low energy content by volume, and high content of oxygenated compounds, which damage combustion

producing prelycopaoctaene diphosphate (PLPP), which rearranges into

contains β-1,4 and/or β-1,3 glucans making a cellulose-like polymer [20].

plasticity of L race to synthesize squalene and lycopadiene.

neering of botryococcene biosynthesis.

Microalgae - From Physiology to Application

metabolism [46].

2.3 Biosynthesis of lycopadiene

not yet identified (Figure 4b).

and p-ferulic acids [48].

3.1 Hydrocarbons

128

diphosphates as substrates [15] (Figure 4).

2.4 Extracellular matrix (ECM) polymers

3. Profitability of B. braunii derivatives

engines and cause corrosion, erosion, and accumulation of deposits in the nozzles; because of these reasons, they are mixed with standard fuels [49, 50]. B. braunii accumulates hydrocarbons similar to those of the crude oil, and their direct contribution in the formation of oil reserves currently in use has been reported [3–5]. The B. braunii oils showed almost equal values in density and surface tension than the diesel, but with higher kinematic viscosity and distillation temperature [50]. The B. braunii race B oil was already converted into diesel with an 85% performance, using a simple conversion process under mild conditions of 260°C and 1 atm. The physical properties are relatively close to the specification for diesel, with 40 as estimated cetane (CN) number [51].

The limitation to use B. braunii as biorefinery is the slow growth rate of days in comparison with hours in other algae [49, 52]. Other factors affecting the growth


ASLW, aerated swine lagoon wastewaters (not sterile); °C, temperature; CO2, % v/v; Dt, doubling time (days); NIA, no information available; PAR, photosynthetic active radiation (μmols of photons/m<sup>2</sup> s); Php, photoperiod (light/ dark hours); SCGR, specific cell growth rate (μ/day); μ, specific velocity of growth rate; St, strain (race); THC, total hydrocarbons (% DW, dry weight).

a Blue light <sup>λ</sup> = 470 nm. <sup>b</sup>

W/m<sup>2</sup> . <sup>c</sup>

Estimated values [54].

d Calculated values from μ, using Ln(2)/μ equation.

#### Table 1.

Comparison of culture conditions and productivity of hydrocarbons between B. braunii strains at laboratory scale.

and hydrocarbon production are the strain, CO2, light, water, nutrients, temperature, pH, and salinity [53–55, 60] (Table 1). A JET PASTER treatment was used to do a mechanical cell disruption and removal of the polysaccharides of the B. braunii colonies, increasing the hydrocarbon extraction up to 82.8%. This treatment did not affect the photosynthetic function of the cells [56]. On the other hand, a repetitive nondestructive extraction with heptane was reported as having some advantages [57]. Also, a continuous growth and extraction column of n-dodecane was reported recently as an efficient hydrocarbon extraction method without significant loss of the viability of the cells [58]. Considering these milking procedures and achieving a 10% rate of return, a minimum sales price (MSP) of US\$3.20 per liter was calculated, and a reduction down to US\$1.45 per liter was proposed, if hydrocarbon content increases and extraction procedures become more efficient [59].

3.2 Lipids

UTEX 572 (A)

KMITL 2 (n.d.)

KMITL 2 (n.d.)

LB572 (A)

IBL-C117

LB572 (A)

2441 (A)

131

B. braunii also produces saturated and monounsaturated fatty acids, especially palmitic (16:0) and oleic (18:1), as well as triacylglycerols (TAGs). The percentages of total lipids as saturated, monounsaturated, and polyunsaturated fatty acids in dry biomass are around 44.97, 9.85, 79.61, and 10.54%, respectively [64, 75]. Studies in vitro and in vivo showed that these fatty acids effectively improve the absorption

B. braunii stores TAGs and saturated fatty acids in the lag phase as an adaptation to stress conditions but most are synthesized during the stationary phase. Although highest content of these acids is intracellular, B. braunii secretes oily drops in small

The yield and lipid composition depends on the strain, the culture system used, growth conditions and cell aging, as well as nitrogen, phosphorus, and micronutri-

St System TRT Biomass Lipids Ref.

EF (1 L) 86 mg/L NO3 NIA 0.48 NIA 39.42 0.19 NIA [78]

222 mg/L PO4 NIA 0.86 NIA 54.69 0.47 NIA 444 mg/L PO4 NIA 1.91 NIA 23.23<sup>a</sup> 0.45 NIA 27 mg/L Fe NIA 0.22 NIA 34.93 0.08 NIA

SCGR XMax Px CNT Yld. Prod.

0.04 mM NO3 0.09 0.16 NIA 63 NIA 0.009 [77] 0.37 mM NO3 0.185 0.38 NIA 36 0.19 0.019

0.17 g/L NO3 0.045 4.84 NIA 35.24 NIA 0.016 [79] 2.5 g/L NO3 0.049 5.62 NIA 38.60 NIA 0.0189

Photoaut. (CO2) 0.093 1.14 NIA 25.1 NIA 0.0241 [81] Heterot. (gluc 5 g/L) 0.115 1.75 NIA 29.3 NIA 0.0467

EF (1 L) Chu (0.75) 0.13 0.9 0.12 47.1 NIA NIA [82]

EF (1 L) Chu (0.75) 0.15 1.3 0.18 20.2 NIA NIA [82]

Chu (1.0) 0.13 0.7 0.1 46 NIA NIA Chu (2.0) 0.11 1 0.15 41.3 NIA NIA

Chu (1.0) 0.16 1.4 0.2 22.5 NIA NIA Chu (2.0) 0.17 1.5 0.22 11 NIA NIA

(N:P = 1:1) in Chu NIA 4.963 0.173 33.7 NIA NIA [83] (N:P = 3:3) in Chu NIA 3.857 0.215 34.6 NIA NIA (N:P = 6:6) in Chu NIA 3.987 0.223 32.1 NIA NIA

NIA NIA 0.296 64.96 NIA 0.19 [80]

NIA NIA 0.304 59.56 NIA 0.18

0.195 2.46 NIA 37.5 NIA 0.0645

of lipophilic drugs like flurbiprofen, through the skin [76].

The Colonial Microalgae Botryococcus braunii as Biorefinery

DOI: http://dx.doi.org/10.5772/intechopen.88206

quantities observed on the surface of the cell apex [64].

0083 g/L PO4 and 0.1 g/ L SO4

0058 g/L PO4 and 0.09 g/L SO4

Mixot. (gluc 5 g/L + CO2)

ent concentrations (Table 3).

EF (125 mL)

Outdoor oval pond (150 L)

FBR column (625 mL)

mL)

FBR Airlift (2 L)

TRG EF (250

There are different open and closed culture systems in photobioreactors (PBR) [63, 64], but more studies are required at pilot and industrial scale, to reduce problems by contamination and low yield of biomass and hydrocarbon production [49]. Table 2 summarizes some data about cell growth and hydrocarbon productivity using different culture systems.


°C, temperature; CNT, content (% DW dry weight); CO2, % v/v; HCs, hydrocarbons; PAR, photosynthetic active radiation (μmols of photons/m<sup>2</sup> s); PBR, photobioreactor; Php, photoperiod (light/dark hours); Px, biomass productivity (mg/L day); NIA, no information available; Rcwy, raceway; rT, room temperature; SCGR, specific cell growth rate (μ/day); μ, specific velocity of growth rate; Sol r, solar radiation; St, strain (race); WHC, weight of hydrocarbons (mg/L day); Xmax, maximum cellular concentration (g/L). <sup>a</sup>

"Tickle film" (30.5 16.5 in) continuous. <sup>b</sup> "Airlift" (10 L).

c Panel (1000 L) outdoor and semicontinuous.

d

"Biofilm" (0.275 m<sup>2</sup> or 600 mL). <sup>e</sup>

"Attached" bioreactor (0.08 m<sup>2</sup> or 240 mL). <sup>f</sup>

(25 m<sup>2</sup> or 5000 L) semicontinuous. <sup>g</sup> Estimated values [64].

h g/m<sup>2</sup>

. <sup>i</sup> g/m<sup>2</sup> /day; shadow area indicates the highest reported values up to now.

#### Table 2.

Comparison of culture conditions and productivity of hydrocarbons between strains of B. braunii in bioreactors.
