3.2 Lipids

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].

There are different open and closed culture systems in photobioreactors (PBR)

St System Cultures Biomass HCs Ref

GUBIOT JTBB1 Plain (3 L) 25 35 (16 h) 0% 0.112 NIA 13 52.6 6.8 [62]

Showa (B) PBRa 25-28 282 (15 h) 5–7% NIA 20 1500 22.5 225-340 [68]

LB572 (B) PBR<sup>c</sup> 20 Sol r 0% 0.04 0.3 15 NIA 2.4 [71]

FACHB 357 (B) Attchd<sup>e</sup> 25 500 (24 h) 1% NIA 62<sup>h</sup> 5.5–6.5i 19.43 1.06<sup>i</sup> [73] TN101 Rcwy sc<sup>f</sup> rT Sol r 0% NIA NIA 33.8<sup>i</sup> 22.6 8.2-13<sup>i</sup> [74] °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

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

radiation; St, strain (race); WHC, weight of hydrocarbons (mg/L day); Xmax, maximum cellular concentration (g/L). <sup>a</sup>

29 Sol r 5 kWh/ m2 .day

trophic

°C PAR CO2 SCGR Xmax Px CNT WHC

25 150 (24 h) 20% 0.13g NIA 92.4 24.45g 22.6 [64]

rT Sol r 0% NIA NIA 77.8 19 13.2 [70]

rT Sol r 0% NIA NIA 40 24 10.8 [70]

PBRd 25 55 (24 h) 1% NIA 96.4 0.71i NIA NIA [72]

NIA 4.55 234 29.7 71.1 [69]

0% 0.38 NIA 114 11 12.5 [67]

[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 produc-

tivity using different culture systems.

Microalgae - From Physiology to Application

(3 L)

Circular (50 L)

(80 L)

(1800 L)

NIA PBR<sup>b</sup> 25 270 (24 h) Mixo-

765 Column

N-836 (B) Rcwy

AP103 Rcwy

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

"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].

Panel (1000 L) outdoor and semicontinuous.

/day; shadow area indicates the highest reported values up to now.

UTEX-LB 572 (A)

UTEX-LB 572 (A)

"Airlift" (10 L).

c

d

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

130

Table 2.

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

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

The yield and lipid composition depends on the strain, the culture system used, growth conditions and cell aging, as well as nitrogen, phosphorus, and micronutrient concentrations (Table 3).



promote antioxidant and anti-inflammatory activities [96]. The extracellular polysaccharides (exopolysaccharides, EPS) constitute most of the organic material of high molecular weight released to the environment by microalgae and other microorganisms. They have antioxidant, immunomodulatory, antibacterial, antiviral, anticarcinogenic, and antihypocholesterolemic effects [97]. They are used as thickeners, emulsifiers, bioflocculants, stabilizers, and gelling agents in foods and cosmetics; are soluble in water; and modify the rheological properties of solutions

The ECM and the fibrillar pod are composed of mucilaginous polysaccharides [20], and other detected EPS are fucose, glucose, mannose, rhamnose, uronic acids, and unusual sugars such as 3-O-methyl fucose, 3-O-methyl rhamnose, and 6-O-methyl hexose [1]. Galactose is involved in the innate and adaptive immune system [99]. L-Arabinose is used as food additive for its sweet taste and poor absorption in humans [100] and is an antiglycemic agent by selective inhibition of invertases, reducing the glycemic response after sucrose ingestion [101]. Uronic acid is a chelating agent to remove metal ions. Fucose has high commercial value for its anticancer properties and for chemical synthesis of flavoring

Some B. braunii (UC 58) strains produce 4.0–4.5 g/L EPS with few hydrocarbons (5%). The EPS amount varies with the strain, race, physiological conditions, and culture. Strains of A and B races can produce up to 250 mg/L EPS, and race L up to 1

Greater EPS production correlates with minor growth by N deficiency. Urea and ammonia decrease the pH, as well as EPS production. Optimal conditions for EPS production were nitrate (8 mM) and between 25 and 30°C. Out of these temperatures, the EPS polymerization decreased significantly [1, 102]. Light/dark (16:8) photoperiod produced more hydrocarbons, but continuous light with agitation increased EPS until 1.6 and 0.7 g/L in LB 572 and SAG-30 strains, respectively [103]. EPS production increased (2–3 g/L) in low salinity levels (17–85 mM) as osmoprotectants [53]. High salinity and low N content in D medium induced EPS production (0.549 0.044 g/L) in comparison to the BG11 medium (0.336 0.009 g/L), but biomass was higher in BG11 (1.019 0.051 g/L) than in D (0.953 0.056 g/L) [104]. Modification of culture conditions could be used to increase EPS production, to facilitate the removal, and to increase hydrocarbon recovery. With Botryococcus braunii CCALA 778 (race A), a light:dark cycle at 26°C resulted in an increased production of EPS, and a milking procedure for these polysaccharides has been proposed [105, 106]. EPS can be used as thickening

Algenanes are aliphatic, nonhydrolyzable, and insoluble biopolymers found in the ECM at 9 and 10% dry weight of race A and B, respectively. Due to their high resistance to degradation, they are attributed to the good preservation of colonies in

Another reported biopolymer was the polyhydroxybutyrate (PHB), a biodegradable plastic with a yield of about 20% of the dry weight [109]. PHB is a polyester with thermoplastic and biodegradable properties, and it's a carbon and energy storage compound. For its similar physical properties to polypropylene and polystyrene, it is of commercial interest [110]. Under pH 7.5, 40°C, and with 60% wastewater as culture medium, a maximum yield of 247 0.42 mg/L PHB was

increasing their viscosity to form gels [1, 98].

The Colonial Microalgae Botryococcus braunii as Biorefinery

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

agents [1, 55].

g/L plus glucose [1].

or gelling agents [107].

3.5 Other biopolymers

sedimentary rocks [108].

reported [111].

133

CNT, content (% DW dry weight); Chu, Chu media for microalgae [8]; EF, Erlenmeyer flask; FBR, photobioreactor; N:P, proportion of nitrogen: phosphate; Px, biomass productivity (g/L day); NIA, no information available; Prod., productivity (g/L day); Rcwy, raceway; SCGR, specific cell growth rate (μ/day); μ, specific velocity of growth rate; St, strain (race); TRT, treatment; Yld., yield (g/L); Xmax, maximum cellular concentration (g/L). <sup>a</sup> mg/cm2 . <sup>b</sup>

mg/cm2 /day.

#### Table 3.

Comparison of crop conditions and lipid productivity in B. braunii.

#### 3.3 Pigments

Algae pigments have been reported to have antioxidant, anticancer, antiinflammatory, antiobesity, and antiangiogenic properties and function as neuroprotectives [85]. So, they could replace synthetic dyes in food, cosmetic, nutraceutical, and pharmaceutical products [86].

Carotenoid pigments are unsaturated hydrocarbons, while xanthophylls have one or more functional groups containing oxygen such as lutein, canthaxanthin, and astaxanthin [85–87].

Carotenoids abound in races B and L, lutein being the main pigment (22–29%), followed by others as β-carotene, echinenone, 3-OH echinenone, canthaxanthin, violaxanthin, loroxanthin, and neoxanthin. Transition to stationary phase causes a color change in B. braunii from green to brown, reddish orange, and pale yellow by accumulation of carotenoids and a decrease of intracellular pigments [88]. Canthaxanthin (46%) and echinenone (20–28%) are predominant in the stationary phase in response to nitrogen deficiency [36]. The BOT-20 strain showed a dark red color during growth because of the accumulated echinenone of about 30.5% dry weight and 630 mg/L production, but with few hydrocarbons (8%) [89].

Adonixanthin was detected in race L during the stationary phase [90], and botryoxanthin A, botryoxanthin B, and braunixanthin 1 and 2 were detected in race B [37, 38, 91]. The 2-azahypoxanthine (AHX) similar to the phytohormone induced the accumulation of secondary carotenoids like botryoxanthin A and braunixanthin 1 and decreased the content of botryococcenes during the stationary phase [92], imitating a lack of nitrogen condition without inhibiting the growth.

In race A, lutein (79–84%) is the main carotenoid followed by β-carotene (1.75– 2.14%), violaxanthin (6–9%), astaxanthin (3–8%), and zeaxanthin (0.32–0.78%). In salinity and high light intensity conditions, the lutein increases [53, 93]. All of these compounds shown antioxidant properties and inhibitory effect against lipid peroxidation in vitro and in vivo and activated antioxidant enzymes such as catalase [94, 95].

#### 3.4 Polysaccharides

The aqueous extracts of B. braunii (strain LB 572) reduce the skin dehydration, stimulate collagen synthesis, promote the differentiation of adipocytes, and

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

promote antioxidant and anti-inflammatory activities [96]. The extracellular polysaccharides (exopolysaccharides, EPS) constitute most of the organic material of high molecular weight released to the environment by microalgae and other microorganisms. They have antioxidant, immunomodulatory, antibacterial, antiviral, anticarcinogenic, and antihypocholesterolemic effects [97]. They are used as thickeners, emulsifiers, bioflocculants, stabilizers, and gelling agents in foods and cosmetics; are soluble in water; and modify the rheological properties of solutions increasing their viscosity to form gels [1, 98].

The ECM and the fibrillar pod are composed of mucilaginous polysaccharides [20], and other detected EPS are fucose, glucose, mannose, rhamnose, uronic acids, and unusual sugars such as 3-O-methyl fucose, 3-O-methyl rhamnose, and 6-O-methyl hexose [1]. Galactose is involved in the innate and adaptive immune system [99]. L-Arabinose is used as food additive for its sweet taste and poor absorption in humans [100] and is an antiglycemic agent by selective inhibition of invertases, reducing the glycemic response after sucrose ingestion [101]. Uronic acid is a chelating agent to remove metal ions. Fucose has high commercial value for its anticancer properties and for chemical synthesis of flavoring agents [1, 55].

Some B. braunii (UC 58) strains produce 4.0–4.5 g/L EPS with few hydrocarbons (5%). The EPS amount varies with the strain, race, physiological conditions, and culture. Strains of A and B races can produce up to 250 mg/L EPS, and race L up to 1 g/L plus glucose [1].

Greater EPS production correlates with minor growth by N deficiency. Urea and ammonia decrease the pH, as well as EPS production. Optimal conditions for EPS production were nitrate (8 mM) and between 25 and 30°C. Out of these temperatures, the EPS polymerization decreased significantly [1, 102]. Light/dark (16:8) photoperiod produced more hydrocarbons, but continuous light with agitation increased EPS until 1.6 and 0.7 g/L in LB 572 and SAG-30 strains, respectively [103]. EPS production increased (2–3 g/L) in low salinity levels (17–85 mM) as osmoprotectants [53]. High salinity and low N content in D medium induced EPS production (0.549 0.044 g/L) in comparison to the BG11 medium (0.336 0.009 g/L), but biomass was higher in BG11 (1.019 0.051 g/L) than in D (0.953 0.056 g/L) [104]. Modification of culture conditions could be used to increase EPS production, to facilitate the removal, and to increase hydrocarbon recovery. With Botryococcus braunii CCALA 778 (race A), a light:dark cycle at 26°C resulted in an increased production of EPS, and a milking procedure for these polysaccharides has been proposed [105, 106]. EPS can be used as thickening or gelling agents [107].
