François Gelin

Microalgal biomarkers: A review of recent research developments

Abstract Microalgae are major sources of lipids in lacustrine and marine environments. This paper provides a review of some recent advances in our knowledge of the wide variety of lipid types that have been isolated from microalgae with an emphasis on those likely to be useful biomarkers for identifying sources of organic matter in sediments. Extensive data are now available on the fatty acids in all of the major classes of microalgae and some useful characteristic features have been observed in the abundance of particular polyunsaturated fatty acids. Despite several decades of study, it is now apparent that some of the biosynthetic steps leading to the formation of these unsaturated fatty acids are still not known with certainty as shown by the occurrence of C 28 polyunsaturated fatty acids in some dinoflagellates and the likely involvement of chain-shortening reactions. Considerable data have also been obtained on the sterols in microalgae, but some classes of organisms are still not well documented (e.g. cryptomonads, eustigmatophytes, xanthophytes and raphidophytes). Diatoms show a great variety of sterol compositions and no sterol appears to be either unique or representative. However, 24-methylenecholesterol in sediments is probably derived in most cases from diatoms. High contents of C 25 highly branched isoprenoid (HBI) alkenes have been identified in the diatom Haslea ostrearia and both C 25 and C 30 HBI alkenes have been found in diatom strains thought to be Rhizosolenia setigera . Genetic and environmental factors appear to be important controls on the relative abundances of the various homologues identified. Microalgae are also suspected to be a source of long-chain saturated fatty acids having an even carbon number predominance and of long-chain alkanes with no odd over even carbon number predominance, although the available data are not conclusive. An exciting development in recent years is the identification of highly aliphatic biopolymers (algaenans) in some species of marine and freshwater green algae and eustigmatophytes. This material persists in sediments and may be a source of the alkyl chains in ancient kerogens and crude oil constituents. Algaenans do not occur in all algal species and may be absent from some classes, such as diatoms. This implies that the organic matter preserved in sediments is strongly influenced by a subset of the microalgal contributors of organic matter. Although reasonable sources have been identified for many of the lipids in sediments, there are still many gaps in our knowledge and further studies are clearly required.

Distribution of aliphatic, nonhydrolyzable biopolymers in marine microalgae

The recent discovery of several marine microalgal species which biosynthesize nonhydrolyzable, aliphatic biomacromolecules, termed algaenans, has provided new insights about the nature and origin of the organic matter preserved in marine sediments. This paper reports the examination of 12 new species of marine microalgae from five different classes; the results are discussed together with previous observations on marine algaenans. None of the diatoms and prymnesiophytes and only one of the dinoflagellates examined in the present study produced algaenan, whereas all the eustigmatophytes biosynthesize a very specific algaenan composed of polyether-linked long-chain alkyl units. Many of the chlorophytes investigated, as with their freshwater counterparts, also produce algaenan. The location of the algaenan in the algal cells and the resistance of these materials to chemical and bacterial attack are discussed with respect to their role as a potential sink of organic carbon in aquatic environments.

Resistant biomacromolecules in marine microalgae of the classes Eustigmatophyceae and Chlorophyceae: Geochemical implications

Non-hydrolysable macromolecular constituents (i.e. algaenans) were isolated from two out of seven marine microalgae investigated. Nannochloropsis salina and Nannochloropsis sp. from the class of Eustigmatophyceae produce highly aliphatic algaenans. Flash pyrolysis and chemical degradations with HI and RuO4 allowed for the identification of their chemical structure, which is mainly composed of polyether-linked long-chain (up to C36) n-alkyl units. The building blocks of this polymer were also recognized in lipid fractions. The green microalgae (Chlorophyceae) Chlorella spaerckii, Chlorococcum sp. and Nannochloris sp. were earlier thought to biosynthesize algaenans comprising aliphatic and/or aromatic moieties. However, a new isolation method utilizing trifluoroacetic acid (TFA) prior to the other hydrolyses revealed that the macromolecular material isolated from these three chlorophytes was either hydrolysable with TFA or artefacts from the former method. Similar to algaenans from fresh water green microalgae, the aliphatic eustigmatophyte algaenans are likely to be selectively preserved in depositional environments and might ultimately serve as source rock organic matter of marine crude oils. Furthermore, they may play an important role in the cycling of carbon.

Novel, resistant microalgal polyethers: An important sink of organic carbon in the marine environment?

Abstract Five out of seven marine microalgal species investigated were found to biosynthesize nonhydrolysable, mainly aliphatic, biomacromolecules (algaenans). The molecular structure of the algaenan isolated from the microalga Nannochloropsis salina of the class Eustigmatophyceae was determined by solid state 13 C NMR spectroscopy, Curie point pyrolysis-gas chromatography-mass spectrometry, and chemical degradations with HI and RuO 4 . The structure is predominantly composed of C 28 -C 34 linear chains linked by ether bridges. The algaenan isolated from a second eustigmatophyte ( Nannochloropsis sp.) was structurally similar. Algaenans isolated from two chlorophytes also possess a strongly aliphatic nature, as revealed by the dominance of alkenes/alkanes in their pyrolysates. Accordingly, we propose that the aliphatic character of numerous Recent and ancient marine kerogens reflects selectively preserved algaenans and that these algaenans may act as a source of n -alkanes in marine crude oils.

Mid-chain hydroxy long-chain fatty acids in microalgae from the genus Nannochloropsis☆

Abstract Extractable and bound lipids of four species of microalgae from the genus Nannochloropsis have been examined by capillary GC-mass spectrometry. In addition to previously described unusual C28C34 alkyl diols and n-alcohols, small quantities of compounds identified as C28-C34 monohydroxy fatty acids were detected both in free and bound form. For each carbon chain-length member of the series, a single positional isomer predominated, which was identified as the ω18-isomer from characteristic mass spectral fragment ions. The position of the hydroxyl group at ω18 rather than at a constant position relative to the carboxylic acid group indicates that the series results from chain-elongation (or perhaps chain-shortening) of a particular hydroxy fatty acid, rather than hydroxylation of a range of fatty acids. Furthermore, two dihydroxy fatty acids, identified as 15,16-dihydroxydotriacontanoic and 16,17-dihydroxytritriacontanoic acids, were also found in the products from acid hydrolysis of the cell residue. Possible biosynthetic correlations between these hydroxy and dihydroxy fatty acids and the long-chains diols also produced by these algae are discussed. Our data suggest that hydroxy acids may be precursors of the diols via reduction of the acid group. However, another pathway must be invoked in order to explain the formation of some diol isomers.

The similarity of chemical structures of soluble aliphatic polyaldehyde and insoluble algaenan in the green microalga Botryococcus braunii race A as revealed by analytical pyrolysis

Curie point pyrolysis-gas chromatographic-mass spectrometric studies of a recently isolated CHCl3-soluble aliphatic polyaldehyde, and of an insoluble biopolymer termed Bb(A) algaenan, of the green microalga Botryococcus braunii (race A) were performed to determine the structural relationships between these two polymers. Comparisons of specific mass chromatograms, and of the composition of three clusters of pyrolysis products, with different chain lengths of pyrolysates of both materials clearly show major similarities in their chemical structures. According to these results, the molecular structure of Bb(A) algaenan is probably a more condessed and/ora reticulated form of the soluble aliphatic polyaldehyde. Furthermore, the results confirm that kerogens derived from these algae consist of mixtures of algaenan and polyaldehydes which have become insoluble due to oxygen cross-linking.

Structural characterization of aliphatic, non-hydrolyzable biopolymers in freshwater algae and a leaf cuticle using ruthenium tetroxide degradation

Aliphatic, non-hydrolyzable biopolymers were subjected to RuO4-oxidation in order to examine the potential of this method in revealing details on their structures. The method was tested on model compounds first and found to cleave alkyl chains of aromatic moieties, double bonds and ether bonds. Oxidation of the biopolymer cutan derived from cuticles of Agave americana and the algaenans isolated from the cell walls of the freshwater algae, Botryococcus braunii (race A and L) and Tetraedron minimum, resulted in the formation of specific oxidation products. In the case of B. braunii, the results confirmed previously suggested structures. Results obtained for cutan and T. minimum algaenan enabled, for the first time, the reconstruction of the building blocks of these biopolymers. These blocks consist of unsaturated long-chain hydroxy fatty acids, which are cross-linked via ether-bonds or ester-bonds.

scope and limitations of flash pyrolysis-gas chromatography/mass spectrometry as revealed by the thermal behaviour of high-molecular-weight lipids derived from the green microalga Botryococcus braunii

Abstract Curie point pyrolysis—gas chromatography/mass spectrometry studies of four types of high-molecular-weight (HMW) lipids isolated from the green microalga Botryococcus braunii race A were performed to determine the thermal behaviour of these lipids and to propose mechanisms of pyrolysis for these types of compounds. Although two types of lipids induced detectable pyrolysis products upon heating of the ferromagnetic wires at Curie temperatures of 610 and 770°C, transfer problems from the pyrolysis unit to the GC column were observed. Therefore, further analysis of the pyrolysis residues is suggested. Furthermore, two types of lipids presenting long alkyl chains (up to C64) did not pyrolyse under the experimental conditions but were thermally extracted from the wire at any of the tested temperatures. Some of these HMW lipids could, however, be analysed by high-temperature gas chromatography (temperatures up to 375°C). Mechanisms of pyrolysis, partly based on previous studies, were proposed for the two types of ether lipids. These mechanisms allowed the structural reconstruction of the main biopolymer that composes the cell outer walls of the microalga B. braunii race L. This biomacromolecule was found to be comprised of C40 isoprenoid (lycopene) units, ether linked at the C(14) and C(15) positions.

Mechanisms of flash pyrolysis of ether lipids isolated from the green microalga Botryococcus braunii race A

Two types of ether lipids isolated from the microalga Botryococcus braunii have been subjected to flash pyrolysis. The pyrolysis products were separated and analyzed by GC/MS. The nature and distribution of the pyrolysis compounds gave clues to the different mechanisms involved in the pyrolysis of ether-linked alkyl chains. The relatively abundant presence of alkenes, alkadienes, alken-9-ones and alken-lo-ones with chain lengths corresponding to those of the ether-bond alkyl chains indicated that the cleavage of the C-O bond is an important first step in the pyrolysis process.

Alternative biological sources for 1,2,3,4-tetramethylbenzene in flash pyrolysates of kerogen

Pyrolysates of kerogens isolated from Indian Ocean surface sediments and from Black Sea Unit II contain abundant 1,2,3,4-tetramethylbenzene (1,2,3,4-TMB) suggesting the presence of macromolecularly-bound isorenieratane skeletons derived from isorenieratene biosynthesized by green sulphur bacteria (cf. Hartgers et al., 1994c). However, the 13C content of 1,2,3,4-TMB is identical to that of algal lipids, excluding an origin from green sulphur bacteria. The presence of 1,2,3,4-TMB in pyrolysates of stable residues of several marine algae indicates that it can also have an algal origin. Therefore, the assessment of photic zone anoxia on the basis of the abundant presence of 1,2,3,4-TMB in the pyrolysate is not possible without determination of its stable carbon isotopic composition and comparison with those of algal lipids.