C. Berkaloff

Sites of accumulation and composition of hydrocarbons in Botryococcus braunii

Abstract Raman spectrometry and electron microscopy show that, in the hydrocarbon-rich alga Botryococcus braunii , hydrocarbons accumulate in two distinct sites; internally in cytoplasmic inclusions and externally in successive outer walls and derived globules. No other classes of lipid are present in noticeable amounts in the cytoplasmic inclusions and in the external globules. The same hydrocarbons are observed in the internal and external pools but with different relative abundances, the shorter hydrocarbons being more abundant in the internal pool. The bulk of B. braunii hydrocarbons ( ca 95%) is located in the external pool. Such an extracellular location allows this species to exhibit both an unusually high hydrocarbon content (15% of dry wt) and a normal level (0.75%) within the cells. The hydrocarbon pattern and location of B. braunii were compared with that of other organisms; a close relation appears between higher plant epidermal cells and this green alga. The trilaminar outer walls of B. braunii , at whose contact external hydrocarbon globules accumulate, contain a sporopollenin-like compound.

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.

Structure and chemistry of a new chemical race of Botryococcus braunii (chlorophyceae) that produces lycopadiene, a tetraterpenoid hydrocarbon

New strains of the hydrocarbon rich alga Botryococcus braunii Kützing were isolated from water samples collected in three tropical freshwater lakes. These strains synthesize lycopadiene, a tetraterpenoid metabolite, as their sole hydrocarbon. The morphological and ultrastructural characteristics of these algae are similar to those reported for previously described strains which produce either alkadienes or botryococcenes. The pyriform shaped cells are embedded in a colonial matrix formed by layers of closely appressed external walls: this dense matrix is impregnated by the hydrocarbon and some other lipids. We believe the new strains synthesizing lycopadiene form a third chemical race in B. braunii, besides the alkadiene and botryococcene races, rather than a different species.

Non-hydrolysable macromolecular constituents from outer walls of Chlorella fusca and Nanochlorum eucaryotum

Abstract Many green microalgae possess a thin trilaminar outer wall (TLS) with a very high resistance to chemical degradation. TLS are known to play an important protective role in living cells. They are also selectively preserved during fossilization and thus provide a major contribution to the fossil organic matter of a number of sedimentary rocks. However, little information is available on TLS chemical structure. Examination of the TLS of Chlorella fusca (a lacustrine Chlorophycea) and of Nanochlorum eucaryotum (a recently discovered marine Chlorophycea) indicated that (i) they exhibit morphological features commonly observed in other green microalgae, (ii) their non-hydrolysable macromolecular constituents comprise large amounts of long, up to C 30 , polymethylenic chains, (iii) the absence of carotenoid moieties indicates that they can no longer be considered as sporopollenins and (iv) the large contribution of hydrocarbon chains in these macromolecules accounts for the important role of fossil TLS in the formation of numerous oil-source rocks.

Resistant biopolymer in the outer walls of Botryococcus braunii, B race

Abstract A biopolymer highly resistant to non-oxidative treatments (PRB B ) occurs in the outer walls of the B race of Botryococcus braunii . PRB B accounts for ca 10% of the total algal biomass and fulfils the usual requirements to be regarded as a sporopollenin; however, the structural information obtained indicate it does not derive from carotenoids. A complete lack of relationship was also noted between PRB B and the isoprenoid hydrocarbons produced in large amount by the B race. The PRB B structure is based upon saturated, normal, hydrocarbon chains up to C 31 ; these chains are probably linked by ether bridges. PRB B also comprises ester and hydroxyl groups protected from external attacks by the polymeric network. PRB B and PRB A (the resistant biopolymer isolated from the outer walls of the A race of B. braunii , which produces non-isoprenoid hydrocarbons) are not identical. Nevertheless they show the same major structural features and belong to the samegroup of resistant biopolymers (probably derived from the polymerisation of very long chain fatty acid derivatives). Analogies between PRB A and PRB B would have resulted in the formation, via fossilization of A and B races, of kerogens with a similar structure and a high oil potential.

Occurrence of a resistant biopolymer in the L race of Botryococcus braunii

Abstract A resistant biopolymer, PRB L , occurs in the outer walls of the L race of Botryococcus braunii . It accounts for a larger fraction of the total biomass than PRB A and B do in the A and B races. The PRB L structure is mainly based on long (up to C 30 saturated, isoprenoid hydrocarbon chains probably linked by ether bridges. Most of these isoprenoid chains exhibit regular head-to-tail linkages. Therefore, PRB L does not belong to the same family as PRB A and B , the structures of which are based on long, normal hydrocarbon chains. The only hydrocarbon produced by the L race algae is a lycopadiene so a relationship between hydrocarbon and PRB structure can be considered in this L race as has already been demonstrated for the A race.

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.

Occurrence and origin of «ultralaminar» structures in «amorphous» kerogens of various source rocks and oil shales

Abstract Forty low maturity, type I–II kerogens from source rocks and oil shales, ranging in age from Infra-Cambrian to Miocene, were examined by transmission electron microscopy (TEM). When previously observed by light microscopy and/or UV fluorescence microscopy the above samples, like a very large number of kerogens, seemed homogeneous and amorphous. In fact, TEM revealed the presence of “ultralaminar” structures in 22 of these “amorphous” kerogens. The abundance of “ultralaminae” depends on the considered sample and some of the tested kerogens are nearly exclusively composed of “ultralamina” accumulations. Based on thickness, three main groups of “ultralaminae” can be identified. Parallel studies on extant algae indicated that some of the above “ultralaminae” likely derived from the resistant outer walls of microalgae. The fossilization of such outer walls should occur via the “selective preservation” pathway and result in the formation of highly aliphatic “ultralaminar” kerogens. The lack of “ultralaminae” in Infra-Cambrian kerogens is consistent with a cyanobacterial origin.

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.

First example of an algaenan yielding an aromatic-rich pyrolysate. Possible geochemical implications on marine kerogen formation

Non-hydrolysable macromolecules were isolated from the marine green microalga Chlorella marina where they represent ca. 5% by weight of the dry biomass. Such resistant biomacromolecules, termed algaenans, were previously identified in numerous microalgae (mostly comprising freshwater species) and they were shown to play a major role in the formation of a number of source rocks and oil shales via the selective preservation pathway. Scanning and Transmission Electron Microscopy revealed that the algaenan of C. marina is located in the cell wall but is completely amorphous once isolated. All the previously studied algaenans were also cell wall constituents but they always retained some morphological features of the initial walls. Another conspicuous difference was also observed regarding chemical structure. In agreement with spectroscopic observations, the pyrolysate of C. marina algaenan is sharply dominated by compounds comprising various aromatic units whereas all the so far studied algaenans appeared highly aliphatic and based on long polymethylenic chains. As a result, the selective preservation of algaenans could not be restricted to the formation of aliphatic sedimentary organic matter. In fact, it can be anticipated that some aromatic fractions of kerogens should also be derived from such a pathway, as suggested in recent studies.