Diversos produtos tópicos são desenvolvidos a fim de se evitar os efeitos nocivos da radiação ultravioleta (UV), dentre eles, os protetores solares constituídos por filtros físicos e/ou químicos. No entanto, os filtros químicos podem promover eventos adversos, razão pela qual a busca de ativos em fontes naturais tem-se mostrado de grande importância. A microalga Botryococcus braunii pode ser de potencial interesse em aplicações cosméticas, pois foi descrito que seu extrato aquoso apresentou redução da desidratação cutânea, induziu a produção do colágeno, bem como promoveu atividade antioxidante. Assim, o estudo visou analisar o auxilio da B. braunii na fotoproteção cutânea e na proteção da pele por meio da ação antioxidante in vitro. As formulações, constituídas de biomassa da microalga ou de seus extratos, acrescidas ou não de filtros, foram avaliadas quanto à: ação antioxidante, atividade fotoprotetora in vitro e fotoestabilidade. A B. braunii apresentou bom desempenho de crescimento, contendo lipídeos (25,65%) e proteínas (49,17%) em proporção semelhante à descrita na literatura. A presença de fenóis totais e a ação antioxidante foram analisadas nos extratos obtidos com clorofórmio, metanol e etanol 70%. O extrato obtido com etanol 70% foi o que apresentou maior concentração de fenóis totais (68,9 µg/mL de ácido cafeico), porém inferior ao relatado na literatura; e a ação antioxidante não foi considerada significativa ao se comparar com o padrão Trolox®. Nas formulações, os extratos foram considerados ineficientes para ação antioxidante. Quanto à atividade fotoprotetora in vitro e fotoestabilidade, a B. braunii não apresentou eficácia adequada, existindo, porém, a possibilidade de os extratos atuarem na região da luz visível e contribuírem contra os efeitos danosos provocados por esta radiação à pele. / Several topical products are developed in order to avoid the harmful effects of ultraviolet (UV) radiation, including sunscreens made by physical and/or chemical filters. However, chemical filters may promote adverse events, which is why the search for actives from natural sources has proven to be of great importance. The microalgae Botryococcus braunii may be of potential interest in cosmetic applications, because it has been reported that its aqueous extract showed a reduction in skin dehydration, induced collagen production and promoted antioxidant activity. Thus, the study aimed to analyze the contribution of B. braunii in skin photoprotection and skin protection through in vitro antioxidant action. The formulations, consisting of biomass from the microalgae or their extracts, supplemented or not by filters, were evaluated for: antioxidant activity, photoprotective activity in vitro and photostability. The B. braunii showed good growth performance, containing lipids (25.65%) and protein (49.17%) in similar proportion to the described by the literature. The presence of phenolic compounds and antioxidant activity were analyzed in extracts obtained with chloroform, methanol and 70% ethanol. The extract obtained with 70% ethanol showed the highest concentration of total phenolics (68.9 mg/mL caffeic acid), but lower than that reported in the literature; and the antioxidant action was not considered significant when compared to the Trolox® standard. In the formulations, the extracts were considered inefficient for antioxidant action. In relation to the in vitro photoprotection activity and photostability, B. braunii did not provide adequate efficacy, however, there is a possibility that the extracts have an ability to act in the visible light region and contribute to the protection from the harmful effects caused by that radiation to the skin.

As microalgas são organismos unicelulares, eucariotos, simples em estrutura, fotossintetizantes, que requerem principalmente luz, água e nutrientes inorgânicos para seu crescimento. Estes micro-organismos podem converter eficientemente dióxido de carbono em biomassa e certos compostos bioativos com aplicações nas indústrias alimentícia, farmacêutica e cosmética. Botryococcus braunii é uma espécie de microalga capaz de sintetizar e armazenar lipídios, incluindo ácidos graxos, epóxidos, alquilfenol, éter e hidrocarbonetos de cadeia longa. O conteúdo lipídico pode alcançar de 15% a 75% da biomassa seca, sendo que os ácidos graxos livres representam mais da metade deste extrato. Os parâmetros como carbono inorgânico, nitrogênio, vitaminas e sais são vitais para o crescimento de microalgas, exercem influência tanto no crescimento como na composição da biomassa. Assim, este trabalho tem a finalidade de verificar a influência das quantidades de nitrato de sódio e de fosfato de potássio dibásico no crescimento e composição da microalga Botryococcus braunii, onde serão avaliados parâmetros cinéticos e de crescimento, bem como a composição da biomassa. Os diferentes meios de cultivo avaliados para o crescimento da microalga mostraram a importância da influência da salinidade no crescimento e composição da biomassa de B. braunii, sendo o meio Chu encontrado como o melhor meio para o crescimento, no qual para uma otimização do crescimento e produção de biomassa foram utilizadas diferentes concentrações de fontes de nitrogênio e fósforo. Com uso de meio Chu, foi possível a obtenção de concentrações celulares de 4962,9mg.L-1 em fotobiorreator tubular, com correção da concentração das fontes de nitrogênio e fósforo de acordo com o crescimento celular. Nas várias concentrações de nitrogênio e fósforo avaliadas, foi encontrada uma concentração de lipídios numa faixa de 32,56 a 36,93%, onde foram encontrados os seguintes ácidos graxos C11:0; C14:1; C16:0; C16:1; C17:1; C18:0; C18:1n9; C18:2n6; C20:0; C18:3n6; C20:1 e C18:3n3. / Microalgae are unicellular photosynthetic organisms, eukaryotes, simple in structure, requiring mainly light, water and inorganic nutrients for growth. These micro-organisms can efficiently convert carbon dioxide into biomass and certain bioactive compounds with applications in food, pharmaceutical and cosmetic industries. Botryococcus braunii is a microalga capable of synthesizing and storaging lipids, including fatty acids, epoxides, ether, and hydrocarbons. Lipid content can reach 15% to 75% of dry matter, and free fatty acids represent more than half of this extract. Parameters such as inorganic carbon, nitrogen, vitamins and salts are important parameters regulating algal growth and the composition of biomass. This work aims to study the influence of the amounts of sodium nitrate and potassium phosphate dibasic in the growth and composition of microalgae Botryococcus braunii, by evaluating kinetic and growth parameters, as well as biomass composition. The culture media evaluated for the growth of microalgae, showed the importance of the influence of salinity on growth and biomass composition of Botryococcus braunii. Chu medium was found as the best medium for growth, for which an optimization of growth and biomass concentrations different sources of nitrogen and phosphorus was used. Using the Chu medium, it was possible to obtain cell concentrations of 4962.9mg.L-1 in tubular phobioreactor, with correction of concentrations of both nitrogen and phosphorus sources according to cell growth. Taking into account the various nitrogen and phosphorus evaluated, A concentration of lipids was found in a range from 32.56 to 36.93%, where the fatty acids are: C11:0; C14:1; C16:0; C16:1; C17:1; C18:0; C18:1n9; C18:2n6; C20:0; C18:3n6; C20:1 and C18:3n3.

Diversos produtos tópicos são desenvolvidos a fim de se evitar os efeitos nocivos da radiação ultravioleta (UV), dentre eles, os protetores solares constituídos por filtros físicos e/ou químicos. No entanto, os filtros químicos podem promover eventos adversos, razão pela qual a busca de ativos em fontes naturais tem-se mostrado de grande importância. A microalga Botryococcus braunii pode ser de potencial interesse em aplicações cosméticas, pois foi descrito que seu extrato aquoso apresentou redução da desidratação cutânea, induziu a produção do colágeno, bem como promoveu atividade antioxidante. Assim, o estudo visou analisar o auxilio da B. braunii na fotoproteção cutânea e na proteção da pele por meio da ação antioxidante in vitro. As formulações, constituídas de biomassa da microalga ou de seus extratos, acrescidas ou não de filtros, foram avaliadas quanto à: ação antioxidante, atividade fotoprotetora in vitro e fotoestabilidade. A B. braunii apresentou bom desempenho de crescimento, contendo lipídeos (25,65%) e proteínas (49,17%) em proporção semelhante à descrita na literatura. A presença de fenóis totais e a ação antioxidante foram analisadas nos extratos obtidos com clorofórmio, metanol e etanol 70%. O extrato obtido com etanol 70% foi o que apresentou maior concentração de fenóis totais (68,9 µg/mL de ácido cafeico), porém inferior ao relatado na literatura; e a ação antioxidante não foi considerada significativa ao se comparar com o padrão Trolox®. Nas formulações, os extratos foram considerados ineficientes para ação antioxidante. Quanto à atividade fotoprotetora in vitro e fotoestabilidade, a B. braunii não apresentou eficácia adequada, existindo, porém, a possibilidade de os extratos atuarem na região da luz visível e contribuírem contra os efeitos danosos provocados por esta radiação à pele. / Several topical products are developed in order to avoid the harmful effects of ultraviolet (UV) radiation, including sunscreens made by physical and/or chemical filters. However, chemical filters may promote adverse events, which is why the search for actives from natural sources has proven to be of great importance. The microalgae Botryococcus braunii may be of potential interest in cosmetic applications, because it has been reported that its aqueous extract showed a reduction in skin dehydration, induced collagen production and promoted antioxidant activity. Thus, the study aimed to analyze the contribution of B. braunii in skin photoprotection and skin protection through in vitro antioxidant action. The formulations, consisting of biomass from the microalgae or their extracts, supplemented or not by filters, were evaluated for: antioxidant activity, photoprotective activity in vitro and photostability. The B. braunii showed good growth performance, containing lipids (25.65%) and protein (49.17%) in similar proportion to the described by the literature. The presence of phenolic compounds and antioxidant activity were analyzed in extracts obtained with chloroform, methanol and 70% ethanol. The extract obtained with 70% ethanol showed the highest concentration of total phenolics (68.9 mg/mL caffeic acid), but lower than that reported in the literature; and the antioxidant action was not considered significant when compared to the Trolox® standard. In the formulations, the extracts were considered inefficient for antioxidant action. In relation to the in vitro photoprotection activity and photostability, B. braunii did not provide adequate efficacy, however, there is a possibility that the extracts have an ability to act in the visible light region and contribute to the protection from the harmful effects caused by that radiation to the skin.

As microalgas são organismos unicelulares, eucariotos, simples em estrutura, fotossintetizantes, que requerem principalmente luz, água e nutrientes inorgânicos para seu crescimento. Estes micro-organismos podem converter eficientemente dióxido de carbono em biomassa e certos compostos bioativos com aplicações nas indústrias alimentícia, farmacêutica e cosmética. Botryococcus braunii é uma espécie de microalga capaz de sintetizar e armazenar lipídios, incluindo ácidos graxos, epóxidos, alquilfenol, éter e hidrocarbonetos de cadeia longa. O conteúdo lipídico pode alcançar de 15% a 75% da biomassa seca, sendo que os ácidos graxos livres representam mais da metade deste extrato. Os parâmetros como carbono inorgânico, nitrogênio, vitaminas e sais são vitais para o crescimento de microalgas, exercem influência tanto no crescimento como na composição da biomassa. Assim, este trabalho tem a finalidade de verificar a influência das quantidades de nitrato de sódio e de fosfato de potássio dibásico no crescimento e composição da microalga Botryococcus braunii, onde serão avaliados parâmetros cinéticos e de crescimento, bem como a composição da biomassa. Os diferentes meios de cultivo avaliados para o crescimento da microalga mostraram a importância da influência da salinidade no crescimento e composição da biomassa de B. braunii, sendo o meio Chu encontrado como o melhor meio para o crescimento, no qual para uma otimização do crescimento e produção de biomassa foram utilizadas diferentes concentrações de fontes de nitrogênio e fósforo. Com uso de meio Chu, foi possível a obtenção de concentrações celulares de 4962,9mg.L-1 em fotobiorreator tubular, com correção da concentração das fontes de nitrogênio e fósforo de acordo com o crescimento celular. Nas várias concentrações de nitrogênio e fósforo avaliadas, foi encontrada uma concentração de lipídios numa faixa de 32,56 a 36,93%, onde foram encontrados os seguintes ácidos graxos C11:0; C14:1; C16:0; C16:1; C17:1; C18:0; C18:1n9; C18:2n6; C20:0; C18:3n6; C20:1 e C18:3n3. / Microalgae are unicellular photosynthetic organisms, eukaryotes, simple in structure, requiring mainly light, water and inorganic nutrients for growth. These micro-organisms can efficiently convert carbon dioxide into biomass and certain bioactive compounds with applications in food, pharmaceutical and cosmetic industries. Botryococcus braunii is a microalga capable of synthesizing and storaging lipids, including fatty acids, epoxides, ether, and hydrocarbons. Lipid content can reach 15% to 75% of dry matter, and free fatty acids represent more than half of this extract. Parameters such as inorganic carbon, nitrogen, vitamins and salts are important parameters regulating algal growth and the composition of biomass. This work aims to study the influence of the amounts of sodium nitrate and potassium phosphate dibasic in the growth and composition of microalgae Botryococcus braunii, by evaluating kinetic and growth parameters, as well as biomass composition. The culture media evaluated for the growth of microalgae, showed the importance of the influence of salinity on growth and biomass composition of Botryococcus braunii. Chu medium was found as the best medium for growth, for which an optimization of growth and biomass concentrations different sources of nitrogen and phosphorus was used. Using the Chu medium, it was possible to obtain cell concentrations of 4962.9mg.L-1 in tubular phobioreactor, with correction of concentrations of both nitrogen and phosphorus sources according to cell growth. Taking into account the various nitrogen and phosphorus evaluated, A concentration of lipids was found in a range from 32.56 to 36.93%, where the fatty acids are: C11:0; C14:1; C16:0; C16:1; C17:1; C18:0; C18:1n9; C18:2n6; C20:0; C18:3n6; C20:1 and C18:3n3.

Botryococcus braunii is a microscopic, colonial green alga that may be found in fresh and brackish waters throughout the globe. B. braunii is unique in that it constitutively synthesises and secretes copious amounts of various long-chain (C23-C40) hydrocarbons, generically termed “botryococcenes”. Botryococcanes, the hydrogenated forms of botryococcenes, comprise 1% of the fossil hydrocarbons found in petroleum deposits and in oil-shales. Microfossils identified as Botryococcus by optical and scanning electron microscopy are also abundant in these strata, but the actual identity and precise relationship between these microfossils and extant Botryococcus species is not known. In this investigation, the relationship between living Botryococcus algae and microfossils identified as Botryococcus using traditional palaeontological analysis and light-microscopy was investigated by analysis of ancient DNA (aDNA). The material used was identified in sediments from Boswell Lake (British Columbia, Canada), a Holocene lake that had remained undisturbed since the glacial retreat. New flow-cytometry methods were developed to rapidly purify enough of the relevant microfossils, from which aDNA was extracted and sequenced. Pollen grains were purified using the same flow-cytometry method and from the same horizons as the Botryococcus microfossils and used to age the sedimentary horizons by 14C radiocarbon dating. Samples of the purified microfossils were imaged by scanning electron microscopy for comparison with published images of fossils identified as Botryococcus from kerogens. In addition, metaDNA from the relevant horizons was extracted and sequenced by NGS, and a chemical analysis for botryococcene derivatives performed using two-dimensional gas chromatography (2D-GC). The genomic analyses show that the sub-fossils identified in Boswell Lake are likely to be representatives of B. braunii, race B. The geochemical analysis identified hydrocarbons that migrate as botryococcenes on 2D-GC in the strata whence the sub-fossils were purified. The SEM images indicate that the microfossils purified from Boswell Lake have similar morphologies to those found in kerogens. Taken together, these data strongly support the proposition that petroleum and kerogen deposits are unusually rich in B. braunii and that these algae have a lineage potentially dating 500 million years. The metagenomic analysis enabled similar conclusions to be reached regarding the presence of B. braunii within the sediment, without the need for targeted microfossil purification. While this analysis was less precise due to the under-representation of algal genomes in the public sequence databases, the metagenomics approach employed was particularly well suited to the temporal analysis of prokaryotic microcosms within Lake Boswell, the succession of which could be associated with periods of climatic variation. The analytical methods described herein are generally applicable to understanding microbial systems over geological periods, and may be used to generate important insights into the cause and effect relationships between microbial populations and environmental perturbation.

Difficulties associated with the study of microbial communities, such as low proportions of cultivable species, have been addressed in recent years with the advent of a range of sequencing technologies and bioinformatic tools. This is enabling previously unexplored communities to be characterised and utilised in a range of biotechnology applications. In this thesis bioinformatic methods were applied to two datasets of biotechnological interest: microbial communities found living with the oil-producing alga Botryococcus braunii and microbial communities in acid mine drainage (AMD). B. braunii is of high interest to the biofuel industry due to its ability to produce high amounts of oils, in the form of hydrocarbons. However, a number of factors, including low growth rates, have prevented its cultivation on an industrial scale. Studies show B. braunii lives in a consortium with numerous bacteria which may influence its growth. This thesis reports both whole genome analysis and 16S rRNA gene sequence analysis to gain a greater understanding of the B. braunii bacterial consortium. Bacteria have been identified, some of which had not previously been documented as living with B. braunii, and evidence is presented for ways in which they may influence growth of the alga, including B-vitamin synthesis and secretion systems. AMD is a worldwide problem, polluting the environment and negatively impacting on human health. This by-product of the mining industry is a problem in the South West of England, where disused metalliferous mines are now a source of AMD. Bioremediation of AMD is an active area of research; sulphur-reducing bacteria and other bacteria which can remove toxic metals from AMD can be utilised for this purpose. Identifying bacteria and archaea that are able to thrive in AMD and which also have these bioremediation properties is therefore of great importance. Metagenomic sequencing has been carried out on the microbial community living in AMD sediment at the Wheal Maid tailings lagoon near Penryn in Cornwall. From these data have been identified a diverse range of bacteria and archaea present at both the sediment surface level and at depth, including microorganisms closely related to taxa reported from metalliferous mines on other continents. Evidence has been found of sulphur-reducing bacteria and of pathways for various other bioremediation-linked processes.

B. braunii is a green alga that has attracted attention as a potential renewable fuel source due to its high oil content and the archeological record of its unique contribution to oil and coal shales. Three extant chemotypes of B. braunii have been described, namely race A, race B, and race L, which accumulate alkadienes and alkatrienes, botryococcene and squalene and their methylated derivatives, and lycopadiene, respectively. The methylated triterpenes, particularly botryococcenes, produced by race B can be efficiently converted to high quality combustible fuels and other petrochemicals; however, botryococcene biosynthesis has remained enigmatic. It has been suggested that botryococcene biosynthesis could resemble that of squalene, arising from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form pre-squalene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene, or in an alternative reductive rearrangement, botryococcene. Based on the proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence, isolated squalene synthase-like genes from B. braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encode for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether and to a lesser extent squalene, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when SSL-1 is combined with either SSL-2 or SSL-3, in vivo and in vitro, robust squalene or botryococcene biosynthesis was observed, respectively. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved inter-dependently for specialized triterpene production. Expression of various configurations of the SSL genes in TN-7 yeast demonstrates that botryococcene can be efficiently produced in a heterologous host. Additionally, three triterpene methyltransferase (TMTs) were isolated which efficiently catalyze the transfer of a methyl group from S-adenosyl methionine (SAM) to either squalene (TMT-1 and TMT-2) or botryococcene (TMT-3) in vivo and in vitro. Co-expression of the various TMT genes with either squalene synthase or botryococcene synthase in TN-7 yeast resulted in the accumulation of C31 and C32 methyl derivatives of squalene or botryococcene, demonstrating their potential for heterologous production. The methylation sites were determined by NMR spectroscopy to be identical to C31 and C32 methyl-derivatives of squalene or botryococcene observed in B. braunii race B. Expression studies of various heterologous squalene synthase genes in S. cerevisiae corroborated an earlier but surprising observation reported in the literature. While the squalene synthase gene of S. cerevisiae was able to complement an erg9 (squalene synthase) knockout in yeast, squalene synthase genes from plants and animals were not. Chemical profiles revealed that squalene accumulated to significant levels in yeast expressing the squalene synthase of plant, animal, or S. cerevisiae. This suggested that it was not the ability of these heterologous synthase enzymes to produce squalene, but their inability to feed squalene into the native sterol biosynthetic pathway that prevented them from restoring normal ergosterol biosynthesis in S. cerevisiae. By examining the ability of chimera squalene synthase enzymes to complement the erg9 mutation, a discrete sequence of amino acids near the C-terminus of the enzyme was identified which is necessary and sufficient for allowing any squalene synthase to restore normal sterol metabolism.

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 +/- 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 +/- 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 +/- 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600?1700 cm^-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm^-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm^-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells.

Le kérogène, fraction insoluble de la matière organique sédimentaire, est un mélange complexe et hétérogène de macromolécules organiques. Ces structures évoluent, essentiellement sous l’effet de la température, au cours des temps géologiques et génèrent les hydrocarbures présents dans les bassins sédimentaires. Comprendre et quantifier les mécanismes physicochimiques associés à ce processus est important pour l’estimation des réserves pétrolières. Au cours de cette étude, deux géopolymères ont été sélectionnés pour représenter la décomposition thermique de structures typiques des kérogènes naturels. Dans un premier temps, une étude expérimentale nous a permis (1) de proposer des structures moléculaires des géopolymères et (2) de décrire les mécanismes primaires de décomposition des géopolymères. Les échantillons ont été analysés au moyen d’expériences de pyrolyse en milieu confiné à cinq températures de référence comprises entre 200 à 300°C. Un schéma cinétique correspondant aux processus précoces de décomposition a été établi à partir des observations expérimentales pour chacun des deux géopolymères. Dans un deuxième temps, les modèles moléculaires élaborés dans l'étape précédente ont été soumis à des simulations moléculaires dynamiques utilisant un champ de forces réactif (ReaxFF). Ces simulations ReaxFF ont apporté une interprétation théorique aux processus-clés observés expérimentalement. L’ensemble des résultats de cette étude suggère que les modèles cinétiques, en une étape implémentés dans les simulateurs de bassin standard ne reproduisent pas correctement la physicochimie des processus de décomposition de la matière organique dans les roches mères naturelles / Kerogen, the insoluble fraction of sedimentary organic matter, is a complex mixing of organic macromolecules, the structure of which evolves during geological times as a function of temperature mainly. The thermal evolution of kerogen is at the origin of hydrocarbon deposits in sedimentary basins. Understanding and quantifying the physicochemical processes associated to this transformation is therefore important to improve the evaluation of petroleum systems. During this study, two geopolymers were selected in order to represent the thermal decomposition of typical structures in natural kerogen. Firstly, an experimental protocol was set up (1) to define molecular structures of the geopolymers, and (2) to describe primary mechanisms of decomposition of both geopolymers. The two samples were analysed using off-line pyrolysis experiments, at five reference temperatures comprised between 200 to 300°C. A kinetic scheme accounting for early decomposition processes was derived from these experimental observations for each geopolymer. Secondly, molecular models were submitted to molecular dynamic simulations using a reactive force field (ReaxFF). ReaxFF simulations provided theoretical supports to the key-processes derived from laboratory experiments. On the overall, results of this study suggested that kinetic models in one step (= parallels reactions implemented in standard (commercial) basin simulators do not adequately reproduce the physicochemistry of organic matter decomposition processes in natural source rocks

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.