Appl Microbiol Biotechnol (2005) 66: 486���496 DOI 10.1007/s00253-004-1779-z MINI-REVIEW P. Metzger . C. Largeau Botryococcus braunii: a rich source for hydrocarbons and related ether lipids Received: 6 July 2004 / Revised: 23 September 2004 / Accepted: 24 September 2004 / Published online: 4 December 2004 # Springer-Verlag 2004 Abstract This paper presents a review on Botryococcus braunii, a cosmopolitan green colonial microalga charac- terised by a considerable production of lipids, notably hydrocarbons. Strains like wild populations of this alga differ in the type of hydrocarbons they synthesise and accumulate: (1) n-alkadienes and trienes, (2) triterpenoid botryococcenes and methylated squalenes, or (3) a tetraterpenoid, lycopadiene. In addition to hydrocarbons and some classic lipids, these algae produce numerous series of characteristic ether lipids closely related to hydrocarbons. This review covers the algal biodiversity, the chemical structures and biosynthesis of hydrocarbons and ether lipids and the biotechnological studies related to hydrocarbon production. Introduction Botryococcus braunii is a green colonial microalga widespread in freshwater and brackish lakes, reservoirs, ponds, or even ephemeral lakes situated in continental, temperate, alpine, or tropical zones (Wake and Hillen 1980, 1981 Aaronson et al. 1983 Huszar and Reynolds 1997 Huang et al. 1999 Metzger and Largeau 1999 Volova et al. 2003). This alga is characterised by a conspicuous ability to synthesise and accumulate a variety of lipids. These lipid substances include numerous hydrocarbons, i.e. highly reduced compounds comprising only carbon and hydrogen as elements (Brown and Knights 1969 Knights et al. 1970), and a number of specific ether lipids (Metzger et al. 1991 Metzger and Largeau 1999). To avoid any confusion, it is important to specify what kinds of compounds are considered as ���lipids��� in the present Mini-Review. According to the classic definition, lipids are all the compounds produced by living organisms which are sparingly soluble in water but readily soluble in organic solvents (Ratledge and Wilkinson 1988) and their structures may contain straight long hydrocarbon chains or isoprene units and various functional groups, especially oxygenated ones. In this paper, we choose to separate B. braunii lipids by a functional group approach, so that straight-chain and isoprenoid hydrocarbons are presented together, while a second section is devoted to ether lipids. Strains of B. braunii isolated and grown in laboratories and wild populations of this alga both differ in the type of hydrocarbons they synthesise. Accordingly, they are sub- classified into three chemical races. Algae in race A produce essentially n-alkadiene and triene hydrocarbons, odd-carbon-numbered from C23 to C33 (Metzger et al. 1985a), algae in race B produce triterpenoid hydrocarbons, C30���C37 botryococcenes (Metzger et al. 1985a) and C31��� C34 methylated squalenes (Huang and Poulter 1989a Achitouv et al. 2004) and algae in race L produce a single tetraterpenoid hydrocarbon, lycopadiene (Metzger and Casadevall 1987 Metzger et al. 1990). The chemical structures of some characteristic hydrocarbons typical of the three chemical races of B. braunii are shown in Fig. 1. In addition to hydrocarbons, B. braunii also synthesises classic lipids such as fatty acids, triacyl glycerols and sterols and a list of these and their distributions can be found in a previous review (Metzger and Largeau 1999). A second feature of this alga is the production of numerous ether lipids of a new type which are not glycerol derivatives, like those occurring in all other living organisms. In each race, ether lipids are closely related to hydrocarbons and in some strains their production can be largely dominant. Lastly, non-polysaccharide biopolymers of very high molecular weight (104 Da to 4��106 Da), polyaldehydes and polyacetals, have been isolated from lipid extracts of B. braunii. Their occurrence and possible functions as precursors of the insoluble polymeric material P. Metzger (*) . C. Largeau Laboratoire de Chimie Bioorganique et Organique Physique, Ecole Nationale Sup��rieure de Chimie de Paris, 11 Rue Pierre et Marie Curie, 75231 cedex 05 Paris, France e-mail: pierre-metzger@enscp.jussieu.fr Tel.: +33-144-276717 Fax: +33-143-257975

building up the outer walls of the alga were recently reviewed by Metzger and Largeau (2002). Owing to its oil richness and its ability to form blooms, sometimes enduring over many years, like in the Darwin Reservoir in Australia (Wake and Hillen 1980 Townsend 2001), this microalga has been proposed as a renewable source of liquid fuel (Casadevall et al. 1985). Furthermore, oil production via CO2 fixation could also mitigate the emission of greenhouse gases (Pedroni et al. 2001). Several studies were therefore carried out to determine the optimal conditions for B. braunii culture and hydrocarbon production. The present article focusses first on the algal biodiver- sity, the chemical structures of hydrocarbons and ether lipids and their biosynthesis and then on some biotechno- logical developments related to the production of algal hydrocarbons. Biodiversity of B. braunii Mophology and taxonomy Colonies of B. braunii under microscope observation exhibit a typical morphology (Fig. 2a) characterised by a botryoid organisation of individual pyriform-shaped cells held together by a refringent matrix containing lipids. Oil droplets can be excreted from the matrix by the pressure of a coverglass. Ultrastructural studies reveal that the matrix surrounding the basal part of the cells consists of outer walls originating from successive cellular divisions (Fig. 2b). Furthermore, the bulk of B. braunii hydrocarbons are stored in these outer walls (Largeau et al. 1980). However, there exists an important morphological heterogeneity within algae examined after water-sampling from lakes and cultivation of strains in the laboratory. The most striking variations concern the size and shape of cells, which can be more or less embedded in the matrix, and the presence (or not) of refringent threads linking clusters of cells, thus leading to the formation of very large colonies (Fig. 2c). On the basis of such morphological differences, but ignoring chemical analyses, Kom��rek and Marvan (1992) proposed the existence of at least 13 species in Botryococcus. However, Plain et al. (1993) noted that, in each chemical race and for the same strain, some of these features could vary in relation to age and culture conditions. Recently, 18S rRNA sequences of four strains of B. braunii belonging to the three chemical races established that these strains formed a monophyletic group (Senousy 2003 Senousy et al. 2004). Now, whether the numerous strains of B. braunii belong to a single species, to three species in connection with the nature of the synthesised hydrocarbons, or to several sub-species is still under debate. Hydrocarbon content Today, a total of about 60 samples of B. braunii, including strains cultivated in laboratory and wild samples collected from lakes, have been analysed for their hydrocarbon content and composition (for a non-exhaustive list, see Metzger and Largeau 1999). They originate from all climatic zones, with the exception of the Antarctic. While Fig. 1 Types of hydrocarbons produced by the three chemical races of B. braunii (I: Knights et al. 1970 II: Villarreal-Rosales et al. 1992 III: Metzger and Ca- sadevall 1983 IV: Huang and Poulter 1989b V: Metzger and Casadevall 1987) 487