Large amount of drilling waste associated with the expansion of the Orinoco Oil Belt (OOB), the biggest proven reserve of extra-heavy crude oil (EHCO) worldwide, is usually impregnated with EHCO and highly salinized water-based drilling fluids. Oxidative exoenzymes (OE) of the lignin-degrading enzyme system (LDS) of fungi catalyse the oxidation of a wide range of toxic pollutants. However, very little evidences on fungal degradation or biotransformation of EHCO have been reported, which contain high amounts of asphaltenes and its biodegradation rate is very limited. The aims of this work were to study the ability of Pestalotiopsis palmarum BM-04 to synthesize OE, its potential to biotransform EHCO and to survive in extreme environmental conditions. Enzymatic studies of the LDS showed the ability of this fungus to overproduce high amounts of laccase (LACp) in presence of wheat bran or lignin peroxidase (LIPp) with EHCO as sole carbon and energy source (1300 U mgP−1 in both cases). FT-IR spectroscopy with Attenuated Total Reflectance (ATR) analysis showed the enzymatic oxidation of carbon and sulfur atoms in both maltenes and asphaltenes fractions of biotreated EHCO catalysed by cell-free laccase-enriched OE using wheat bran as inducer. UV-visible spectrophotometry analysis revealed the oxidation of the petroporphyrins in the asphaltenes fraction of biotreated EHCO. Tolerance assays showed the ability of this fungus to grow up to 50 000 p.p.m. of EHCO and 2000 mM of NaCl. These results suggest that P. palmarum BM-04 is a hopeful alternative to be used in remediation processes in extreme environmental conditions of salinity and EHCO contamination, such as the drilling waste from the OOB.

Chytridiomycosis is a catastrophic disease currently decimating worldwide amphibian populations, caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. Massive species decline to extinction catalyzes radical changes in ecosystems globally, including the largest continuous rainforest ecosystem on Earth, the Amazon rainforest. Innovative research that aims to propose feasible mechanisms of mitigation and the origins of the disease is vital, including studies addressing climatic effects on the expansion of chytridiomycosis. Thus, this publication aims to provide a comprehensive review of: i) the current technologies used for B. dendrobatidis detection and monitoring, and ii) the known Neotropical amphibian's skin microbiota with anti-fungal properties against B. dendrobatidis. Several immunologic and DNA-based methods are discussed to understand the emerging fungal pathogens and their effects on the biosphere, which can help to mitigate the devastating ecological impacts of mass amphibian morbidity. The establishment of rapid and highly accurate B. dendrobatidis detection techniques and methods for monitoring amphibian's cutaneous microbiome is crucial in the fight against chytridiomycosis.

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El biodiseño y biofabricación de biomateriales a partir de residuos vegetales lignocelulósicos y auto-generados por el micelio de hongos es un campo de investigación emergente desde las últimas dos décadas. Surge una nueva cultura material que se basa en los nuevos paradigmas de la fabricación alternativa partiendo de la lógica “de hacer crecer los nuevos materiales en lugar de extraerlos” e integrando los principios básicos de la economía circular y de la Biotecnología Material, asegurando la susceptibilidad de los mismos a ser biodegradados y volver a su estado original en la naturaleza. Su implementación a nivel industrial en distintas áreas de la manufactura comienza a competir con el cuero de origen animal, materiales y productos de origen petroquímico, a la vez que promueve nuevas alternativas de alimentos proteicos sustentables que contribuyan al cambio de los patrones de consumo humano de alto impacto ambiental arraigados a nivel global. La presente revisión, aborda una mirada particular que va desde lo molecular a lo global sobre la nueva cultura micelial, considerando aspectos generales del reino Fungi, la morfogénesis, composición química e integridad celular del micelio, los sistemas multienzimáticos extracelulares de degradación de lignocelulosa que poseen los hongos, pasando por los principales sustratos empleados, los biomateriales desarrollados a partir de micelio a nivel industrial, destacando los biotextiles, materiales y productos para el empaquetamiento y aislamiento, nuevas fuentes alimentarias basadas en el micelio, el arte y el diseño arquitectónico. Finalmente, se presenta el estado del arte actual de las empresas o laboratorios vanguardistas que suscitan una economía circular basada en el micelio de hongos a nivel mundial, al reemplazar recursos y productos de origen fósil por materiales amigables con el entorno, generando alternativas sostenibles y ciclos de producción con una baja demanda de energía y sin repercusiones al medio ambiente, es decir, promoviendo una nueva conciencia material.

Cas13a from Leptotrichia wadeii (LwaCas13a) will be used for all experiments in this proposal. The LwaCas13a protein is not currently available commercially, but the LwaCas13a protein vector for expression can be found on Addgene (pC013-Twinstrep-SUMO-huLwCas13a, Plasmid #90097) in the Feng Zhang Lab Plasmids section (Gootenberg et al., 2017). A previously published production and purification protocols for LwaCas13a will be followed according to Gootenberg et al., (2017) with some modifications described by Iwasaki and Batey (2020). The pET28-APho plasmid is a vector constructed in our laboratory. The plasmids will be transformed into BL21(DE3) Rosetta Escherichia coli cells. The functional activity of LwaCas13a and APhoproteins will be assessed by a reliable fluorescence signal to detect the activity when Cas13a starts to exert its collateral RNAse activity. The restriction map of the pC013 plasmid and pET28-APho plasmid are shown in the following Figure : For the assembling of the RNP complex, Synthego recommends sgRNA:Cas9 ratios between 3:1 and 9:1 for RNP formation in trasnfection experiments. However, the SHERLOCK system recommends a sgRNA:Cas13a ratio of 1:1. We will start with the ratio recommended by SHERLOCK.