Pierre Metzger

Glacial–interglacial environmental changes inferred from molecular and compound-specific δ13C analyses of sediments from Sacred Lake, Mt. Kenya

Abstract Molecular stratigraphic analyses, including lipid distributions and compound-specific δ13C measurements, have been performed at 15 levels in a sediment core from Sacred Lake, Mt. Kenya, a high-altitude (2350 m a.s.l.) freshwater lake with a record extending from the last glacial (>40,000 cal. yr BP) through the present interglacial. Terrestrial and aquatic organic-matter sources were independently assessed using source-specific biomarkers. δ13C values of long-chain n-alkyl lipids from terrestrial higher plants exhibit large glacial to interglacial shifts: those from the last glacial maximum (LGM) (−20 to −18‰) indicate a terrestrial vegetation dominated by C4 grasses or sedges, whereas those from the early Holocene (−34 to −27‰) reflect recolonization of the catchment area by C3 plants, consistent with a rapid rise in the upper treeline. Specific algal biomarkers, including five unsaturated hydrocarbons of novel structure ascribed to the microalga Botryococcus braunii, were abundant, as confirmed by scanning electronic microscopy (SEM). An extreme δ13C shift of over 25‰ is displayed by the algal biomarkers, an elevated value of −5.1‰ at the last glacial maximum (LGM) contrasting with a minimum value of −30.3‰ at the beginning of the Holocene. A major change in the molecular distributions of the algal biomarkers parallels this large δ13C shift, with acyclic isoprenoid hydrocarbons dominating the last glacial and cyclic isoprenoid hydrocarbons the Holocene. The low atmospheric partial pressure of CO2 (pCO2) at the LGM would favour photosynthetic organisms possessing CO2-concentrating mechanisms, including terrestrial C4 grasses and freshwater green algae. Hence, glacial/interglacial changes in pCO2, and in the CO2:O2 ratio in particular, had a significant impact on both terrestrial and aquatic ecosystems on Mt. Kenya, in addition to the effects of climate and local environmental factors.

Alkadiene- and botryococcene-producing races of wild strains of Botryococcus braunii

Abstract Samples of the green colonial alga Botryococcus braunii, collected from various localities, were grown in the laboratory and examined for their hydrocarbon content and morphology. Although few differences appeared between the ultrastructures of the samples, the nature of their hydrocarbons, which remains unchanged at any stage of growth, allows the distinction of two physiological races viz algae producing odd-numbered unbranched alkadienes and trienes (C25C31) (the A race) and those producing polymethylated triterpenes CnH2n-10 (C30C37), the botryococcenes (the B race). In laboratory culture, the hydrocarbon content of these new strains is very high, from 30 to 60% of the dry biomass. For the two races the greatest hydrocarbon productivity takes place during the active growth phase. The important variability observed in botryococcene distribution could originate both from genetic and environmental factors.

Formation of Botryococcus-derived kerogens—Comparative study of immature torbanites and of the extent alga Botryococcus braunii

The outer walls of the extant alga Botryococcus braunii were shown to contain large amounts of a biopolymer, termed PRB (Polymere Resistant de Botryococcus), characterized by a very high resistance towards chemical attacks. Such features suggested that PRB could play a prominent role in the formation of Botryococcus-derived kerogens. In the present work we examined the structure and the origin of PRB using i.r., 13CNMR, pyrolysis-GC and feeding experiments. It appears that PRB synthesized by the living alga comprises chiefly long unbranched, or weakly branched, hydrocarbon chains (containing some cis unsaturations, OH and ester function) probably linked by ether bridges. This biopolymer derives from the dienic hydrocarbons occuring in high level in the alga, or from some long chain close precursors of the latter. A comparative study of PRB with Coorongite and immature Torbanites (i.r., 13CNMR, H/C and O/C atomic ratios, preliminary results from pyrolysis-GC) indicates that PRB is very likely the major constituent of the initial biomass from which Botryococcus-derived kerogens were built. The biosynthesis of PRB by B. braunii accounts for the efficiency of this alga to produce large amounts of kerogens with a high oil potential.

Lycopadiene, a tetraterpenoid hydrocarbon from new strains of the green alga Botryococcus braunii

Abstract Lycopadiene (2,6R,10R,14,19,23R,27R,31 -octamethyldotriaconta- 14(E), 18(E)-diene) has been isolated from the hexane extract of new strains of the green colonial alga Botryococcus braunii . Synthesized in relatively high amount, up to 8% of dry wt, this tetraterpene could be a precursor of lycopane found in some recent and ancient sediments.

Structures of some botryococcenes: branched hydrocarbons from the b-race of the green alga Botryococcus braunii

Nine branched hydrocarbons of the botryococcene type (CnH2n-10 30 ⩽ n ⩽ 37) have been isolated from the green alga Botryococcus braunii. Hydrocarbon mixtures were recovered from wild algae collected in fresh water lakes or from the same strains growing in laboratory; they were further separated by reversed-phase, and in some cases by normal phase, HPLC. From chemical investigations, GC/MS analyses, 1H and 13C NMR spectroscopy, the structures of four new botryococcenes (one C33H56, two C34H58 and one C37H64) were elucidated.

Similar morphological and chemical variations of Gloeocapsomorpha prisca in Ordovician sediments and cultured Botryococcus braunii as a response to changes in salinity

Abstract Most Ordovician source rocks consist of accumulation of a colonial marine microorganism, Gloeocapsomorpha prisca (G. prisca) whose nature, ecology and affinity with extant organisms have been in dispute for years. Furthermore, recent studies have shown major differences in phenol moieties between two G. prisca-rich samples. Examination of five G. prisca-rich kerogens by electron microscopy and pyrolysis studies revealed (i) the occurrence of two markedly distinct “morpho/chemical” types: a “closed/phenol-rich” type (Baltic samples) and an “open/phenol-poor” one (North American samples) and (ii) the selective preservation of the resistant micromolecular material building up the thick cell walls in the original organism. Comparison with extant Botryococcus braunii (a widespread green microalga) grown on media of increasing salinity suggests that G. prisca is likely to be a planktonic green microalga related to B. braunii, which can adapt to large salinity variations which, in turn, control its polymorphism. The large differences in colony morphology and in the content of phenol moieties observed in fossil G. prisca and the resulting occurrence of two “morpho/chemical” types, should therefore reflect depositional environments with different salinities. The presence of thick, highly aliphatic, resistant walls in G. prisca selectively preserved during fossilization, accounts for the major contribution of this organism to Ordovician organic-rich sediments and for the resulting typical signature of Ordovician oils.

A comparative study of macromolecular substances of a Coorongite and cell walls of the extant alga Botryococcus braunii

A Coorongite sample of Lake Balkash (Kazakhstan, CIS) was analyzed in detail by [sup 13]C-NMR, FTIR, Curie point pyrolysis-gas chromatography-mass spectrometry, and by fractionation and derivatization with dimethyldisulphide of an off-line pyrolysate. Both the spectroscopic and the pyrolysis data indicate that the Coorongite was derived almost entirely of organic matter of the green microalga Botryococcus braunii race A. Homologous series of n-alkanes and n-alk-1-enes in all pyrolysates indicated the presence of algaenan, a highly aliphatic and resistant cell wall biomacromolecule of B. braunii race A. Highly specific pyrolysis products, in particular n-alkadienes, n-alkatrienes, alk-1-en-[omega][sup 9]-ones, and alk-1-en-[omega][sup 10]-ones with C[sub 27], C[sub 29], and C[sub 31] carbon atoms clearly indicated that C[sub 27], C[sub 29], and C[sub 31] alkadienes and alkatrienes, originally present in B. braunii race A as such, were cross-linked by oxygen during the very early stages of diagenesis under oxic conditions. Furthermore, several types of dialkenylethers, also present as soluble lipids in B. braunii race A, had undergone cross-linking by oxygen as well. These cross-linked lipids contribute significantly to the Coorongite and clearly demonstrate that under specific conditions kerogen consists of both preserved biomacromolecules and insoluble, cross-linked, low-molecular-weight lipids. 43 refs., 14 figs., 1 tab.

An n-alkatriene and some n-alkadienes from the A race of the green alga Botryococcus braunii

Abstract The various strains of the hydrocarbon-rich alga Botryococcus braunii (A race) examined up to now produce the Z -isomers of the following dienes: tricosa-1,14-diene, pentacosa-1,16-diene, heptacosa-1,18-diene, nonacosa-1,20-diene and hentriaconta-1,22-diene. A C 29 triene was also detected in these strains, but its structure was not fully elucidated. In the present work, spectroscopic data and oxidative degradations, established the structure of the C 29 trienic hydrocarbon. These techniques were also used to examine the dienic series of a new A strain, collected from nature and laboratory grown. In addition to the usual Z -dienes, E -isomers of tricosa-1,14-diene, pentacosa-1,16-diene, heptacosa-1,18-diene and nonacosa-1,20-diene, were characterized.

Biosynthesis of triterpenoid hydrocarbons in the B-race of the green alga Botryococcus braunii. Sites of production and nature of the methylating agent

Abstract The B race of the green alga Botryococcus braunii is characterized by the production of large amounts of botryococcenes, i.e. triterpenoid hydrocarbons of general formula CπH2π-109 n= 30–37. The axenic strain used in this work produces botryococcenes ranging from C30 to C34 when fast growth is promoted by air-lift. Sequential extraction of hydrocarbons with solvents showed that botryococcenes accumulate in two distinct sites: externally in the successive outer walls forming a dense matrix and internally, probably in cyctoplasmic inclusions. Moreover, chase experiments after feeding the algae with sodium [1,2-14C]acetate, and feeding experiments with L -[Me-14C]methionine established the existence of an excretory process from the cells towards the matrix. The results of the radio GC analyses of the botryococcenes synthesized during the feeding experiments provided good evidence to show that the C30 botryococcene is the precursor of all the higher hydrocarbons, and that each intermediate botryococcene C31-C33 is the precursor of its next highest homologue. L -Methionine acts as the methyl donor in the methylation process, leading from the C30 precursor to the botryococcene family. The 13C NMR spectra of the botryococcenes produced when the algae were fed with L -[Me-13C]methionine indicate that the methylation takes place on the C30 backbone in positions 37, 16 and 20.

Bacterial degradation of green microalgae : incubation of Chlorella emersonii and Chlorella vulgaris with Pseudomonas oleovorans and Flavobacterium aquatile

Abstract The influence of cell wall composition on the bacterial degradation of various constituents of green microalgae was examined during prolonged incubation in the dark with two ubiquitous, aerobic, heterotrophic bacteria ( Pseudomonas oleovorans and Flavobacterium aquatile ). The algae belong to the same genus, Chlorella , and were killed by heat shock prior to incubation. The two species exhibit conspicuous differences in cell wall composition: presence of both a classical polysaccharide wall and of a trilaminar outer wall (TLS) composed of highly aliphatic, non-hydrolysable macromolecules (algaenan) in C. emersonii and lack of such a resistant outer wall in the case of C. vulgaris . The changes induced by the bacteria in the abundance and the distribution of the algal hydrocarbons, fatty acids (FA), triacylglycerols (TAG) and chlorophyll (Chl) were determined after 1 and 4 months of incubation. Transmission and scanning electron microscopy observations showed that the algal cell walls were not disrupted by the initial heat shock. A complete lack of bacterial attachment to (or penetration into) the incubated cells was also noted after four months, indicating that bacterial degradation was probably mediated by extracellular enzymes. Examination of the bacteria-free controls showed large decreases in the algal constituents, especially after 4 months. Such non-bacterial degradation could originate, in the case of the hydrocarbons, FA and TAG, from radical oxidations initiated by the formation of hydroperoxide derivatives of polyunsaturated FA, whereas another type of pathway appeared to be implicated in chlorophyll alteration. Important additional decreases in algal hydrocarbons, FA and TAG, reflecting bacterial attack, were noted in the case of the incubated algae. Due to a combination of non-bacterial and bacterial degradation processes, a sharp lowering in the abundance of all the tested compounds was always observed in the incubation experiments. Moreover, comparison of the TLS-containing and of the TLS-devoid algae did not reveal clear-cut differences in the extent of hydrocarbon, FA, TAG and Chl degradation. Accordingly, no specific protective influence appears to be associated with the presence of an algaenan-containing TLS in C. emersonii . It is well documented that TLS plays a major and direct role in the formation of a number of kerogens from source rocks and oil shales. The present results, in agreement with previous TEM observations on such kerogens, suggest that TLS would not play an additional indirect role, during fossilization, via the protection of diagenetically-sensitive constituents of algal cells.