Abstract

The growing shortage of fossil fuels caused an increase in the demand for alternative and renewable fuels. Biofuels, like bioethanol and biodiesel, have received more attention as a sustainable replacement of fossil fuels. However, these have a poor oxidative stability, little energy content by volume, and many oxygenated compounds, which may cause corrosion and damage to the engines. Therefore, they are used as a mixture with standard fuels. Some species of microalgae are candidates to produce oils as triglycerides (TGA) to produce biodiesel by transesterification; however, the problem will remain. The colonial microalgae Botryococcus braunii produces and accumulates a high amount of long-chain nonoxygenated hydrocarbons, similar to those obtained from the fractionated distillation of crude petroleum. This is one of the few organisms reported to have a direct contribution in the formation of the oil reserves currently in use. Additionally, B. braunii produces pigments and long-chain carbohydrates that have interesting properties for various industries. There are still problems to be solved in order to consider it as economically viable and profitable, but important progress is being made. Therefore, this microalga is very attractive for the synthesis of hydrocarbons and other value-added compounds, making it an interesting biorefinery organism.

Keywords: biorefinery, Botryococcus, exopolysaccharides, hydrocarbons, lipids, pigments

### 1. Introduction

Botryococcus braunii is a colonial microalga Trebouxiophyceae, distributed in brackish and sweet water [1]. It reaches densities of 1.4 <sup>10</sup><sup>6</sup> colonies/L [2], and its geochemistry significance is important. Paleobotanical studies suggest that it is one of the largest sources of hydrocarbons in oil-rich deposits dating back to the Ordovician period [1, 3–5]. It is the only colonial microalga that accumulates and secrets liquid hydrocarbons (Figure 1), and depending on the strain and growing conditions, race B can accumulate hydrocarbons up to 85% and race A up to 61% of their dry weight.

B. braunii is related with Characium vaculatum and Dunaliella parva [1]. Due to the hydrocarbons and the molecular phylogeny of B. braunii [6], it is classified in three races (A, B, and L). Race A produces n-alkadienes and alkatrienes of C23–C33

[7], although two unusual hydrocarbons have been characterized, the triene C27H51 and tetraene C27H48 [1]. Race A hydrocarbon dry weight varies from 0.4 to 61% [7, 8]. Race B produces triterpenoids hydrocarbons known as botryococcenes (CnH2n10, n = 30-37) [9] and methylsqualenes C31–C34 [10, 11]. The botryococcenes can be from 27 to 86% of the dry weight [12]. Race L produces a tetraterpene C40 known as lycopadiene and constitutes from 0.1 to 8% of the dry weight [13, 14]. This race contains 5% of lycopatriene, lycopatetraene, lycopapentaene, and lycopahexaene [15]. In addition, a race S is proposed, which synthesizes saturated n-alkanes C18 and C20, and epoxy-alkanes; however, its existence is not yet fully accepted [6].

After the hydrocracking process and subsequent distillation, race B hydrocarbons become biofuels currently used in internal combustion engines [16] as shown

B. braunii races differ also by its morphological and physiological characteristics.

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

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

Symbiotic bacteria have been reported after microscopic observations, and an ectosymbiont α-proteobacteria (BOTRYCO-2) that promotes the productivity of

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].

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

reproduction mechanism of B. braunii seems to be autosporic [26].

biomass and hydrocarbons was described [2, 27].

2.1 Biosynthesis of alkadienes and alkatrienes

2.2 Biosynthesis of botryococcenes

125

2. Physiology and biochemistry of Botryococcus braunii

The Colonial Microalgae Botryococcus braunii as Biorefinery

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

Cells from A and B races are of 13 μm 7–9 μm, and those of L race are

in Figure 2.

8–9 μm 5 μm [19].

(race B) biosynthesis [23].

Figure 1. B. braunii race B colony secreting liquid hydrocarbons.

#### Figure 2.

Hydrocarbons produced by the B. braunii races. Biofuels derived from race B are shown. RON, research octane number = 92–98, this is a measure of autoignition resistance in a spark-ignition engine. In the USA: regular (97 RON) and premium (95 RON). Adapted from [16–18].

[7], although two unusual hydrocarbons have been characterized, the triene C27H51 and tetraene C27H48 [1]. Race A hydrocarbon dry weight varies from 0.4 to 61% [7, 8]. Race B produces triterpenoids hydrocarbons known as botryococcenes

botryococcenes can be from 27 to 86% of the dry weight [12]. Race L produces a tetraterpene C40 known as lycopadiene and constitutes from 0.1 to 8% of the dry

lycopapentaene, and lycopahexaene [15]. In addition, a race S is proposed, which synthesizes saturated n-alkanes C18 and C20, and epoxy-alkanes; however, its

Hydrocarbons produced by the B. braunii races. Biofuels derived from race B are shown. RON, research octane number = 92–98, this is a measure of autoignition resistance in a spark-ignition engine. In the USA: regular (97

(CnH2n10, n = 30-37) [9] and methylsqualenes C31–C34 [10, 11]. The

weight [13, 14]. This race contains 5% of lycopatriene, lycopatetraene,

existence is not yet fully accepted [6].

Microalgae - From Physiology to Application

B. braunii race B colony secreting liquid hydrocarbons.

RON) and premium (95 RON). Adapted from [16–18].

Figure 1.

Figure 2.

124

After the hydrocracking process and subsequent distillation, race B hydrocarbons become biofuels currently used in internal combustion engines [16] as shown in Figure 2.
