Obtaining Biodiesel by Direct Transesterification of Botryococcus braunii and Coccomyxa subellipsoidea

Biofuels have been obtained from vegetable oils, animal fats and recently from microorganisms such as algae, bacteria or yeasts that present a significant content of triacylglycerols through a transesterification reaction. Technical problems with biodiesel or agrodiesel (Term recently used, in this work we will use biodiesel), include oxidative stability, cold flow, and increased NOx emissions. The solution to these problems involves the use of additives on the one hand or modifying the fatty acid composition of the microorganisms alternatively, either through changes in the cultivation temperature, addition of nanomaterials, or through genetic modification, to obtain high-quality biofuels. In this work, two species of microalgae with high fatty acid content were obtained, Botryococcus braunii originating from Cuzco, Peru and Coccomyxa subellipsoidea C-169, from Marble Point, Antarctica, both were cultivated under optimal conditions and later direct transesterification was performed to obtaining biodiesel. The main objective was to develop an economical and viable process that allows it to compete with fossil fuels, based on obtaining biomass and the transesterification method. Here, direct transesterification was effective and allows to significantly reduce the problems associated with the increase in costs of obtaining biodiesel, because reduces critical steps, also the biodiesel thus obtained represents an advance in the development of high-quality biofuels, large-scale and inexpensive. Resumen. Los biocombustibles se han obtenido desde finales del siglo XIX a partir de aceites vegetales, grasas animales y recientemente a partir de microorganismos como algas, bacterias o levaduras que presentan un contenido significativo en triacilgliceroles mediante una reacción de transesterificación. Algunos de los problemas técnicos con el biodiésel o agrodiesel (término usado recientemente, en este trabajo usaremos biodiesel) son: estabilidad oxidativa, flujo en frío y aumento de emisiones de NOx. La solución a estos problemas incluye el uso de aditivos, por un lado, o modificar la composición de ácidos grasos de los microorganismos por otro, ya sea mediante cambios en la temperatura de cultivo, adición de nanomateriales, o mediante modificación genética, para obtener biocombustibles de alta calidad. En este trabajo se utilizaron dos especies de microalgas con gran contenido en ácidos grasos, Botryococcus braunii originaria de Cuzco, Perú y Coccomyxa subellipsoidea C-169, de Punta Mármol, Antártida, ambas fueron cultivadas en óptimas condiciones y posteriormente se realizó la transesterificación directa para obtener el biodiésel. El principal objetivo fue desarrollar un proceso económico y viable, que permita competir con los combustibles fósiles, a partir de la obtención de biomasa y el proceso de transesterificación. La transesterificación directa fue exitosa y permitió reducir significativamente los problemas asociados con el aumento de costos de obtención de biodiésel, ya que reduce pasos críticos del proceso, además el biodiésel así obtenido representa un avance en el desarrollo de biocombustibles de alta calidad, a gran escala y de bajo costo.

Comparative genomics of Chlamydomonas

Despite its role as a reference organism in the plant sciences, the green alga Chlamydomonas reinhardtii entirely lacks genomic resources from closely related species. We present highly contiguous and well-annotated genome assemblies for three unicellular C. reinhardtii relatives: Chlamydomonas incerta, Chlamydomonas schloesseri, and the more distantly related Edaphochlamys debaryana. The three Chlamydomonas genomes are highly syntenous with similar gene contents, although the 129.2 Mb C. incerta and 130.2 Mb C. schloesseri assemblies are more repeat-rich than the 111.1 Mb C. reinhardtii genome. We identify the major centromeric repeat in C. reinhardtii as a LINE transposable element homologous to Zepp (the centromeric repeat in Coccomyxa subellipsoidea) and infer that centromere locations and structure are likely conserved in C. incerta and C. schloesseri. We report extensive rearrangements, but limited gene turnover, between the minus mating type loci of these Chlamydomonas species. We produce an eight-species core-Reinhardtinia whole-genome alignment, which we use to identify several hundred false positive and missing genes in the C. reinhardtii annotation and >260,000 evolutionarily conserved elements in the C. reinhardtii genome. In summary, these resources will enable comparative genomics analyses for C. reinhardtii, significantly extending the analytical toolkit for this emerging model system.

Symbiont composition of the basidiolichen Lichenomphalia meridionalis varies with altitude in the Iberian Peninsula

Basidiolichens are generally poorly researched because of the very small number of species and their restriction to special niches. Lichenomphalia basidiolichens grow in considerable quantities in arctic and alpine habitats but they are inadequately studied in these habitats in Mediterranean areas. Based on morphological and phylogenetic analyses, we identified the different symbionts of L. meridionalis, collected in localities in Spain at altitudes ranging from 533 to 2200 m above sea level. The present study provides the first molecular data available for L. meridionalis. We found that a microindel of six bp within the nrITS2 could help to discriminate L. meridionalis from other species of the genus. Molecular analyses revealed the existence of two different green algal strains, both belonging to Coccomyxa subellipsoidea, a species shared with other Lichenomphalia lichens. Notably, the two chlorobiont strains associated with L. meridionalis were differentially distributed according to altitude, and samples having one of the two strains consistently also included cyanobacteria.

Identification and Characterization of MiRNAs in Coccomyxa subellipsoidea C-169

Coccomyxa subellipsoidea C-169 (C-169) is an oleaginous microalga which is promising for renewable biofuel production. MicroRNAs (miRNAs), as the pivotal modulators of gene expression at post-transcriptional level, are prospective candidates for bioengineering practice. However, so far, no miRNA in C-169 has been reported and its potential impact upon CO2 supplementation remains unclear. High-throughput sequencing of small RNAs from C-169 cultured in air or 2% CO2 revealed 124 miRNAs in total, including 118 conserved miRNAs and six novel ones. In total, 384 genes were predicted as their potential target genes, 320 for conserved miRNAs and 64 for novel miRNAs. The annotated target genes were significantly enriched in six KEGG pathways, including pantothenate and CoA biosynthesis, C5-branched dibasic acid metabolism, 2-oxocarboxylic acid metabolism, butanoate metabolism, valine, leucine and isoleucine biosynthesis and alpha-linolenic acid metabolism. The miRNAs’ target genes were enriched in lipid metabolism as well as RNA-interacting proteins involved in translation, transcription and rRNA processing. The pioneering identification of C-169 miRNAs and analysis of their putative target genes lay the foundation for further miRNA research in eukaryotic algae and will contribute to the development of C-169 as an oleaginous microalga through bioengineering in the future.

Design of a light-gated proton channel based on the crystal structure of Coccomyxa rhodopsin

The light-driven proton pump Coccomyxa subellipsoidea rhodopsin (CsR) provides—because of its high expression in heterologous host cells—an opportunity to study active proton transport under controlled electrochemical conditions. In this study, solving crystal structure of CsR at 2.0-Å resolution enabled us to identify distinct features of the membrane protein that determine ion transport directivity and voltage sensitivity. A specific hydrogen bond between the highly conserved Arg83 and the nearby nonconserved tyrosine (Tyr14) guided our structure-based transformation of CsR into an operational light-gated proton channel (CySeR) that could potentially be used in optogenetic assays. Time-resolved electrophysiological and spectroscopic measurements distinguished pump currents from channel currents in a single protein and emphasized the necessity of Arg83 mobility in CsR as a dynamic extracellular barrier to prevent passive conductance. Our findings reveal that molecular constraints that distinguish pump from channel currents are structurally more confined than was generally expected. This knowledge might enable the structure-based design of novel optogenetic tools, which derive from microbial pumps and are therefore ion specific.