Producción de biomasa de hongos celulolíticos para la degradación de residuos celulósicos

Tesis Universidad Complutense de Madrid,Facultad de Ciencias Biológicas, Departamento de Microbiología III, leída el 26-04-1991Hemos efectuado estudios para la producción de biomasa de hongos celuloliticos capaces de degradar residuos celulosicos en dos fases. En la primera (producción de biomasa fungica), se ha seleccionado un medio de cultivo, asi como un hongo filamentoso capaz de presentar un buen crecimiento sobre dicho medio de cultivo. Hemos efectuado estudios metabólicos (efecto de la aireación, la fuente de nitrógeno y del ph), así como cinéticos (determinación de la tasa especifica de crecimiento, rendimientos y tasas especificas de consumo de sustratos), sobre el desarrollo de este hongo, en cultivo con agitación orbital, modificando las condiciones para la optimización del proceso, y llevando estas posteriormente a fermentador en cultivo en batch. En la segunda fase del trabajo, y con la biomasa fungica obtenida, se ha llevado a cabo la inducción del complejo celulolitico del hongo elegido, utilizando distintos inductores y pretratamientos para asegurar las condiciones optimas de producción enzimática. Estos estudios se han efectuado primero en agitacion orbital y posteriormente en fermentador en cultivo en batchDepto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

The Biology of Pichia membranifaciens Killer Toxins

The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of strains of Saccharomyces cerevisiae capable of secreting killer toxins, much information has been gained regarding killer toxins and this fact has substantially contributed knowledge on fundamental aspects of cell biology and yeast genetics. The killer phenomenon has been studied in Pichia membranifaciens for several years, during which two toxins have been described. PMKT and PMKT2 are proteins of low molecular mass that bind to primary receptors located in the cell wall structure of sensitive yeast cells, linear (1→6)-β-D-glucans and mannoproteins for PMKT and PMKT2, respectively. Cwp2p also acts as a secondary receptor for PMKT. Killing of sensitive cells by PMKT is characterized by ionic movements across plasma membrane and an acidification of the intracellular pH triggering an activation of the High Osmolarity Glycerol (HOG) pathway. On the contrary, our investigations showed a mechanism of killing in which cells are arrested at an early S-phase by high concentrations of PMKT2. However, we concluded that induced mortality at low PMKT2 doses and also PMKT is indeed of an apoptotic nature. Killer yeasts and their toxins have found potential applications in several fields: in food and beverage production, as biocontrol agents, in yeast bio-typing, and as novel antimycotic agents. Accordingly, several applications have been found for P. membranifaciens killer toxins, ranging from pre- and post-harvest biocontrol of plant pathogens to applications during wine fermentation and ageing (inhibition of Botrytis cinerea, Brettanomyces bruxellensis, etc.)

Phenotypic and transcriptional study of the antimicrobial activity of silver and zinc oxide nanoparticles on a wastewater biofilm-forming Pseudomonas aeruginosa strain

The extensive use of nanoparticles (NPs) in industrial processes makes their potential release into the environment an issue of concern. Ag and ZnO NPs are among the most frequently used NPs, potentially reaching concentrations of 1–4 and 64 mg/kg, respectively, in Wastewater Treatment Plants (WWTPs), with unknown effects over microbial populations. Thus, we examined, in depth, the effect of such NPs on a P. aeruginosa strain isolated from a WWTP. We evaluated the growth, ROS production and biofilm formation, in addition to the transcriptomic response in presence of Ag and ZnO NPs at concentrations potentially found in sewage sludge. The transcriptomic and phenotypic patterns of P. aeruginosa in presence of Ag NPs were, in general, similar to the control treatment, with some specific transcriptional impacts affecting processes involved in biofilm formation and iron homeostasis. The biofilms formed under Ag NPs treatment were, on average, thinner and more homogeneous. ZnO NPs also alters the biofilm formation and iron homeostasis in P. aeruginosa, however, the higher and more toxic concentrations utilized caused an increase in cell death and eDNA release. Thus, the biofilm development was characterized by EPS production, via eDNA release. The number of differentially expressed genes in presence of ZnO NPs was higher compared to Ag NPs treatment. Even though the responses of P. aeruginosa to the presence of the studied metallic NPs was at some extent similar, the higher and more toxic concentrations of ZnO NPs produced greater changes concerning cell viability and ROS production, causing disruption in biofilm development

High potential of Pichia kluyveri and other Pichia species in wine technology

The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non-Saccharomyces yeasts such as Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, and Pichia kluyveri have become popular with regard to winemaking and improved wine quality. For that reason, several manufacturers started to offer commercially available strains of these non-Saccharomyces species. P. kluyveri stands out, mainly due to its contribution to wine aroma, glycerol, ethanol yield, and killer factor. The metabolism of the yeast allows it to increase volatile molecules such as esters and varietal thiols (aroma-active compounds), which increase the quality of specific varietal wines or neutral ones. It is considered a low- or non-fermentative yeast, so subsequent inoculation of a more fermentative yeast such as Saccharomyces cerevisiae is indispensable to achieve a proper fermented alcohol. The impact of P. kluyveri is not limited to the grape wine industry; it has also been successfully employed in beer, cider, durian, and tequila fermentation, among others, acting as a promising tool in those fermentation processes. Although no Pichia species other than P. kluyveri is available in the regular market, several recent scientific studies show interesting improvements in some wine quality parameters such as aroma, polysaccharides, acid management, and color stability. This could motivate yeast manufacturers to develop products based on those species in the near future

Desarrollo de un sistema de gestión y control automatizado del programa de mentoría de la UCM

Los alumnos de nuevo ingreso en la Universidad, principalmente los de primer curso de grado, se encuentran con dificultades adicionales a las propiamente académicas al entrar en el nuevo entorno universitario ya que los mecanismos de organización y funcionamiento difieren considerablemente de los que conocen de su experiencia académica anterior. En este proceso de adaptación al nuevo entorno académico, la orientación y acompañamiento por parte de otros estudiantes que han pasado y superado esa situación recientemente es fundamental para reducir el estrés inicial, mejorar el rendimiento y disminuir el periodo de inseguridad inicial. Este proceso de acompañamiento entre iguales es conocido como mentoría. Se define un programa formal de mentoría entre iguales en la universidad como el proceso de acompañamiento en el que un estudiante de cursos superiores (el mentor) mantiene una serie de reuniones con los nuevos estudiantes (telémacos) para facilitarles su integración al entorno y el desarrollo de la carrera, bajo la supervisión de un coordinador. Como cualquier procedimiento cuyo objetivo es una mejora de la calidad, el sistema de mentoría tiene que ser monitorizado y analizado para evaluar su cumplimiento, su utilidad y el grado de satisfacción tanto de los mentores como de los telémacos. Por este motivo, un sistema ágil y dinámico que gestione la información recabada a lo largo del proceso de mentoría permite corregir las disfuncionalidades que se detecten y, de este modo, mejorar los resultados y el grado de satisfacción con el programa. Dado que el programa de mentoría se desarrolla durante el primer cuatrimestre del curso, las actuaciones que tengan que llevarse a cabo para mejorar el proceso deben realizarse de forma muy dinámica y sin demoras, por lo que es necesario un continuo control de cómo se va desarrollando el programa. En la Universidad Complutense de Madrid (UCM) el programa de mentoría para alumnos de nuevo ingreso comenzó a implantarse de forma global en el curso 2012-13. En el curso 2018-19 participan, de forma voluntaria, veinte facultades de un total de veintiséis. Esta implantación tan extensa del programa ha involucrado durante el curso 2018-19 a más de veinte coordinadores, más de 450 mentores y más de 1500 telémacos. En la fase inicial del programa los coordinadores hacen la selección de los mentores en base a sus capacidades e intereses. A estos mentores se les asignan un grupo de telémacos inscritos de forma voluntaria al programa. En la siguiente fase, los mentores planifican una serie de reuniones, entre cinco y ocho, a lo largo de los primeros meses del curso con su grupo de telémacos para tratar en cada una de ellas una temática de interés para los alumnos de nuevo ingreso. Después de cada una de las reuniones, para valorar la utilidad y funcionamiento del sistema, tanto telémacos como mentores rellenan unas encuestas de satisfacción en las que dan su valoración sobre la reunión realizada, las dificultades encontradas, así como el comportamiento y capacidad del mentor. La información de estas encuestas es procesada por el coordinador del programa en la UCM y los resultados son distribuidos a los coordinadores de los distintos centros para que valoren si es necesario realizar reajustes en el funcionamiento del programa para mejorarlo. Igualmente, a cada mentor se le envía la valoración promedio que realizan sus telémacos de la reunión realizada para que les sirva de incentivo para su mejora en reuniones sucesivas. El alto nivel de participación de la UCM en el programa de mentoría, con un gran número de telémacos y mentores, hace que el volumen de datos generado en el proceso de control sea enorme y el análisis de estos datos se ralentiza y dificulta si no se dispone de un sistema automatizado que procese la información. Una obtención y un análisis lentos de los resultados de seguimiento del proceso impediría una intervención ágil y dinámica que se adaptase a las necesidades tanto de mentores como de telémacos. En este proyecto se presenta un sistema de gestión automatizada de los datos basado en herramientas Google, que permite el análisis y distribución de la información en tiempo real y de forma dinámica, facilitando el éxito del programa de mentoría y aumentando el grado de satisfacción de las personas participantes en él

Microbial communities Present in hydrothermal sediments from Deception Island,

Deception Island is a geothermal location in Antarctica that presents active fumaroles, which confers unique characteristics to this habitat. Several studies about microbial communities in Antarctica have been carried out, nevertheless, Antarctic microbiota is still partially unknown. Here we present a multidisciplinary study about sediments obtained by deposition during 4 years in which several approaches have been considered for their characterization. First, a physicochemical characterization, using ionic chromatography and mass spectrometry for the determination of most abundant ions (chloride and sulphate) and elements (mainly silicon), was conducted. In addition, the total microbial community was studied using a metataxonomical approach, revealing a bacterial community dominated by Proteobacteria and Thaumarchaeota as the main archaeal genera and a fungal community mainly composed by Aspergillaceae. Culture-dependent studies showed low microbial diversity, only achieving the isolation of Bacillus-related species, some of them thermophilic, and the isolation of common fungi of Aspergillus or Penicillium spp. Furthermore, diatoms were detected in the sediment and characterized attending to their morphological characteristics using scanning electron microscopy. The study reveals a high influence of the physicochemical conditions in the microbial populations and their distribution, offering valuable data on the interaction between the island and water microbiota

The Genus Metschnikowia in enology

Over the last decade, several non-Saccharomyces species have been used as an alternative yeast for producing wines with sensorial properties that are distinctive in comparison to those produced using only Saccharomyces cerevisiae as the classical inoculum. Among the non-Saccharomyces wine yeasts, Metschnikowia is one of the most investigated genera due to its widespread occurrence and its impact in winemaking, and it has been found in grapevine phyllospheres, fruit flies, grapes, and wine fermentations as being part of the resident microbiota of wineries and wine-making equipment. The versatility that allows some Metschnikowia species to be used for winemaking relies on an ability to grow in combination with other yeast species, such as S. cerevisiae, during the first stages of wine fermentation, thereby modulating the synthesis of secondary metabolites during fermentation in order to improve the sensory profile of the wine. Metschnikowia exerts a moderate fermentation power, some interesting enzymatic activities involving aromatic and color precursors, and potential antimicrobial activity against spoilage yeasts and fungi, resulting in this yeast being considered an interesting tool for use in the improvement of wine quality. The abovementioned properties have mostly been determined from studies on Metschnikowia pulcherrima wine strains. However, M. fructicola and M. viticola have also recently been studied for winemaking purposes

Niche differentiation drives microbial community assembly and succession in full-scale activated sludge bioreactors

Network models and community phylogenetic analyses are applied to assess the composition, structure, and ecological assembly mechanisms of microbial communities. Here we combine both approaches to investigate the temporal dynamics of network properties in individual samples of two activated sludge systems at different adaptation stages. At initial assembly stages, we observed microbial communities adapting to activated sludge, with an increase in network modularity and co-exclusion proportion, and a decrease in network clustering, here interpreted as a consequence of niche specialization. The selective pressure of deterministic factors at wastewater treatment plants produces this trend and maintains the structure of highly functional and specialized communities responding to seasonal environmental changes

Acylase enzymes disrupting quorum sensing alter the transcriptome and phenotype of Pseudomonas aeruginosa, and the composition of bacterial biofilms from wastewater treatment plants

Biofilms represent an essential way of life and colonization of new environments for microorganisms. This feature is regulated by quorum sensing (QS), a microbial communication system based on autoinducer molecules, such as N-acyl-homoserine lactones (AHLs) in Gram negative bacteria. In artificial ecosystems, like Wastewater Treatment Plants (WWTPs), biofilm attachment in filtration membranes produces biofouling. In this environment, the microbial communities are mostly composed of Gram-negative phyla. Thus, we used two AHLs-degrading enzymes, obtained from Actinoplanes utahensis (namely AuAAC and AuAHLA) to determine the effects of degradation of QS signals in the biofilm formation, among other virulence factors, of a Pseudomonas aeruginosa strain isolated from a WWTP, assessing molecular mechanisms through transcriptomics. Besides, we studied the possible effects on community composition in biofilms from activated sludge samples. Although the studied enzymes only degraded the AHLs involved in one of the four QS systems of P. aeruginosa, these activities produced the deregulation of the complete QS network. In fact, AuAAC -the enzyme with higher catalytic efficiency- deregulated all the four QS systems. However, both enzymes reduced the biofilm formation and pyocyanin and protease production. The transcriptomic response of P. aeruginosa affected QS related genes, moreover, transcriptomic response to AuAAC affected mainly to QS related genes. Regarding community composition of biofilms, as expected, the abundance of Gram-negative phyla was significantly decreased after enzymatic treatment. These results support the potential use of such AHLs-degrading enzymes as a method to reduce biofilm formation in WWTP membranes and ameliorate bacterial virulence

Impact of rare yeasts in Saccharomyces cerevisiae wine fermentation performance: Population prevalence and growth phenotype of Cyberlindnera fabianii, Kazachstania unispora, and Naganishia globosa

Saccharomyces cerevisiae is a highly fermentative species able to complete the wine fermentation. However, the interaction with other non-Saccharomyces yeasts can determine the fermentation performance of S. cerevisiae. We have characterised three rare non-Saccharomyces yeasts (Cyberlindnera fabianii, Kazachstania unispora and Naganishia globosa), studying their impact on S. cerevisiae fitness and wine fermentation performance. Using a wide meta-taxonomic dataset of wine samples, analysed through ITS amplicon sequencing, we show that about a 65.07% of wine samples contains Naganishia spp., a 27.21% contains Kazachstania spp., and only a 4.41% contains Cyberlindnera spp; in all cases with average relative abundances lower than 1% of total fungal populations. Although the studied N. globosa strain showed a limited growth capacity in wine, both K. unispora and C. fabianii showed a similar growth phenotype to that of S. cerevisiae in different fermentation conditions, highlighting the outstanding growth rate values of K. unispora. In mixed fermentations with S. cerevisiae, the three yeast species affected co-culture growth parameters and wine chemical profile (volatile compounds, polysaccharides and proteins). K. unispora DN201 strain presents an outstanding capacity to compete with S. cerevisiae strains during the first stage of wine fermentation, causing stuck fermentations in both synthetic and natural grape musts