These are some of the funded projects we are currently working on.

Behaviour and adaptative consequences of bacteria at low growth rates. Andalusian Government, Feder. P20_00774.

We inhabit a microbial planet. Microbial diversity and activity govern numerous limiting processes in nature. Novertheless, there is a large gap of knowledge about the growth rates of microorganisms in nature and the consequences. Our previous results have confirmed a distinctive behaviour of microorganisms in nature and in the laboratory. It is considered that microorganisms live in nature through cycles of feast and famine on their required resources. As well, it is assumed that this lead to the maintenance of the existing large microbial diversity. Thus, microorganisms must experiment periods of growth and maintenance (or near-zero growth) although this is a mostly unknown topic. This study proposes the study of the characteristics of bacterial growth at near-zero rates by comparative analysis to growth under optimum conditions. Different bacterial groups showing distint metabolisms and living strategies will be studied. The full genome sequence of the proposed bacterial species is available which will facilitate further gene expression analyses. The near-zero growth rates will be achieved by using a retentostat, specifically designed to this goal beacuse it represents the only procedure to obtain bacteria at those extremely low growth rates. We propose to decipher if different bacterial groups follow distinctive or similar strategies, the mechanisms at the gene expression level (transcriptomics) that are involved in growth at near-zero growth, and the potential adaptative consequences on the generation of intraspecific heterogeneity at the epigenetic and genomic levels. As a result, this project significantly contributes to understand the world of bacterial growth at highly reduced rates, whether these strategies are species-specific or universal, the mechanisms or strategies of bacterial persistence and to understand the first steps leading to the generation of novel microbial diversity.

Rational design of thermoestable enzymes for saccharification of plant biomass under considerations of transport and accesibility. Andalusian Government, Feder. PY20_RE_021_LOYOLA.

Within the context of modern Biorefineries, this proposal faces processes of valuation of residues from the agricultural industry. Specifically, we will study the optimization of thermoestable enzymes to obtain bioproducts of high added value from residues of rice straw and citric skin. The major difficulty that the processes of enzymatic hydrolysis of plant biomass encounter center around the resistance that it offers to degradation (recalcitrance) by: (I) difficult access of enzymes to glucosidic links and (ii) phenomena of non-productive adsorption and slow kinetics of desorption. We propose modifications in thermophilic enzymes affecting their size and properties of adsorption/desorption to substrates so that the transport and accesibility by enzymes is improved. This study will center of endoglucanases, which decompose the lignocellulosic material to oligosaccharides, products of high added value as prebiotics and promoters of the plant immune system. In summary, the proposal contains the following basic steps: (a) Synthesis of endoglucanases modified to diminish their size and properties of adsoprtion/desorption to the substrate. (b) Evaluation of the enzymatic activity and transport properties of the modified enzymes on plant residues and commercial substrates by comparison to the native enzymes. (c) Development of kinetic models that will contribute to explain the obtained results and will allow redirect future enzymatic manipulations. (d) Initial assays for the development of optimized processes for the conversion of residues on oligosaccharides of medium/high added value. Thanks to the multidisciplinarity of the research team, the project globally faces a process highly transversal containing biochemical interests, chemical and process engineering and physico-chemical and mathematic aspects.

Collaborators: Mauricio Zurita (Universidad de Loyola; Coordinator), Ladero Galan (Universidad Complutense de Madrid), Juan M. González (IRNAS-CSIC)

Fluorescence lifetime spectrometer and its use in Microbiology. Ministerio de Ciencia, Innovación y Universidadesi. Feder. EQC2019-005634-P.

We propose the acquisition of a fluorescence lifetime spectrometer. The equipment will perform TCSPC measurements (Time Correlated Single Photon Counting), a highly sensitive technique which is independent on molecule concentration so that detection and analyses can be carried out in complex samples, and, for example, in cells in vivo. The objective contributes decisively to the field of Microbiology with a powerful tool, so far infra-utilized but with infinite possibilities for analysis and detection. The TCSPC techniques allow to determine specific biomolecules (such as proteins, low molecular weight molecules, probes, etc.) and their interactions with other molecules, under different conditions and as non-destructive method in situ on complex samples both in nature and biotechnology. This will allow the monitorization of prokaryotic physiology and microbial and biotechnological processes. The proposed equipment is able to cover the full spectrum from UV to NIR using liquid or solid samples (including biofilms), and to perform measurements in cuvettes, films, and the quantification in microscope preparations to analyze the physiological state of cells by TCSPC. This equipment will incorporate to the service unit at IRNAS-CSIC Detection and function of microorganisms and their molecules, so that diffusion and use maximizes among all potential users.

Thermophilic Isomerase Processes for Biotechnology. European Union. Horizon 2020. ERA-IB2 7th call. Industrial Biotechnology for Europe. An Integrated Approach. ERA-IB-16-049; Ministerio de Economía y Productividad, Acciones de Programación Conjunta Internacional 2016. PCIN-2016-129.

Isomers are molecules with identical atomic composition but with different structural characteristics. Different isomers can show very distinctive function. Isomerases are enzymes catalyzing the conversion between different types of isomers. Thermostable isomerases are desired because they possess high resistance and durability, are able to withstand harsh industrial process conditions, including heating and organic solvents. Elevated temperatures can also enhance substrate accessibility and solubility. The proposed project includes comparative bioinformatic analyses of sequence data to identify different classes of thermostable isomerases of industrial interest which will be cloned, over-expressed, functionally and structurally characterized and optimized towards their biotechnological application. Three types of isomerases will be targeted: sugar isomerases (to produce new desirable sugars for calorie-free sweeteners and as building blocks for drugs), disulfide isomerases (to improve protein folding and stability of industrial enzymes), and chalcone isomerases (involved in the transformation of flavonoids, secondary metabolites of importance as natural colorants, anti-oxidants, anti-microbial and anti-inflammatory agents). Durable isomerases will allow new opportunities for green, competitive and sustainable biotechnological processes that can replace conventional chemical synthesis.

Collaborators: IRNAS-CSIC (Spain), University of Bergen (Norway), Christian-Albrechts-Universitat Kiel (Germany), University of Exeter (United Kingdom), Bioavan SL (Spain)

Microbial life beyond optimal conditions. Ministry of Economy and Productivity. project CGL2014-58762-P.

Microorganisms are the base to support biogeochemical cycles and terrestrial ecosystems. However, the functioning of microorganisms in natural systems is remains to be deciphered, above all, under conditions considered as extreme or far from the optimum for those cells. It is known that a number of extremophilic microorganisms inhabit soils and sediments although their role has been barely studied. These extremophiles are an ideal target to tackle the role of microorganisms under conditions not included within their usual growth conditions. This proposal is built on the idea that microorganisms are able to perform some activities and maintenance growth at sites with, or during periods of, conditions beyond their usual growth range and the microorganisms in nature behave differently than in laboratory cultures. The actual growth and activity of microorganisms under those unique conditions remains unknown and so are the multiple potential consequences such as explaining high microbial diversity, microbial and enzymatic activities under extreme conditions, cell survival and the interest of applying the knowledge derived from understanding these processes to biotechnology. We propose the analysis of the microbial communities, their enzymatic activity, metabolism, and growth in terrestrial environments using extremophiles as target for this analysis. The use of extremophiles will facilitate the analysis by selecting a specific group of microorganisms from the vast microbial diversity existing in natural environments. Different environmental conditions will be analyzed, including those occurring during the extreme climate events along the year, including summer and winter extremest conditions. Methodologies will include new generation sequencing to characterize the structure of microbial communities under a variety of conditions and their genomic potential based on DNA genomic and amplicon analyses, the identification of metabolically active microbial taxa through their RNA, the determination of enzymatic extracellular activity and microbial metabolism by fluorescence assays and respiration measurements, quantification of growth/death rates through live/dead staining and microscopy and flow cytometer quantification, and evaluation of the potential of these processes and results to biotechnological applications. This study will bring up a significant advance on our understanding of the environmental role of microorganisms and their enzymes under marginalized conditions and it is expected that it will replace the current concept that those situations show minimal relevance. Understanding the behavior of microorganisms under unique conditions will establish a base to apply this knowledge to biotechnology and the sustainability, maintenance and efficient utilization of soils, including global climate changes and the C soil-atmosphere balance.

Effects of water content and temperature on microbial diversity and its activity in soils and sediments. Application to the degradation of halogenated pollutants. Andalusian Government, RNM2529.

Microorganisms in soils and sediments are essential for maintaining these systems. Predictions of global climatic changes and the existence of periods of high temperatures and droughts in our region suggest the importance of knowing the variations in diversity and activity induced on microbial communities in order to achieve an efficient use of resources. These phenomena are critical, for instance, to obtain an appropriate use of soils and sediments for agriculture and the recovery of polluted sites. Microorganisms are the only link able to carry out a high number of processes such as closing the biogeochemical cycles of elements, which are related to the fertilization of terrestrial environments, the degradation of recalcitrant pollutants, and global phenomena. This study will analyze the influence of high temperatures (>40°C) and periods of desiccation on microbial communities, their diversity and activity, in relationship to the decomposition of organic matter and recycling of nutrients in terrestrial environments. This includes their use within a sustainable scheme and the degradation of pollutants under those extreme conditions. To this aim different methods will be used; such as molecular techniques including new generation sequencing to detect the huge microbial diversity existing in soils and sediments, including metagenomics of these natural microbial communities. These methods will be combined with enzyme activity assays that will be designed specifically for these conditions. Experiments will include the analysis of a broad range of temperatures and desiccation conditions both in nature as in the laboratory aiming to understand the functioning and dynamics of microbial communities as a response to changes in water content and temperature, as well as applications to the sustainability of soils and sediments and the recovery of these environments. As an added value, this investigation will search for novel enzymes or biocatalysts which will be characterized. This project represents a collaboration between a research center (IRNAS-CSIC) and a technology-based enterprise (Bioavan SL) which shares a common interest to reach the necessary knowledge to maintain our environment and economic development through sustainability.

Integrative analysis of extremophiles in search for new biotechnological solutions. NILS Science and Sustainability programme, EEA Grants, 003-ABEL-CM-2013.

A sustainable bioindustrial economy requires replacing chemical processes by green, renewable bio-based alternatives. Microorganisms represent the best potential target for biotechnology. Among them, the extremophiles (microorganisms living, for example, under extreme temperature and pH) include the most resistant cells and enzymes required to support the hardiest industrial processes. This action focuses on searching for new hydrolytic biocatalysts, a first step in biotransforming complex residues into assimilable molecules. The strategy includes the major stages: cells, genomes, enzymes and mathematics as an integral approach to model, design and discover novel biocatalysts such as highly resistant enzymes or novel functionality for existing genes. This project involves four research groups, one Norwegian (University of Bergen) and three Spanish teams (IRNAS-CSIC, Center of Astrobiology-CSIC, University of Sevilla). The aim is to join multidisciplinary views to generate biotechnological solutions and advancement into the hydrolysis of cellular materials and residues.

Comparative Microbial Genomics. MICROGEN. Ministry of Science and Innovation, project CSD2009-00006.

Microorganisms are still the main cause of death in the world, they are the main source of genetic biodiversity and the main players in ecosystem functioning. However, the way bacterial populations are structured, the way they evolve or interact with other biotic components or the way they adapt to the environment and evolve are still poorly known. This is partly due to the need for pure culture in order to perform experiments and to the application of an evolutionary model imported from eukaryotic systems. With the advent of genomics and the new high-throughput sequencing techniques of low cost, a new era has emerged in which bacteria can be studied through their genomes, obviating the need for culturing the microorganisms and allowing a holistic study of ecosystems. In addition, the study of this vast genetic reservoir may provide access to a wealth of new bioactive compounds previously unreachable by standard techniques. Some important changes in our way of understanding microbes are starting to emerge, a crucial one being the existence of a gigantic pool of genes within each bacterial species, in such a way that this gene pool (the “pan-genome”) is much larger than the genome of each individual strain. This is vital to understand bacterial biology and has important consequences from an applied point of view.

The present project aims to study microorganisms by a combination of genomics, bioinformatics, molecular biology, metagenomics and next generation sequencing. This will be achieved by the creation of a multidisciplinary team combining all those skills in an ambitious proposal that would not be possible by standard funding. The consortium will use many different bacterial species as models of pathogenicity and symbiosis in human, animal and plant hosts, as well as studying different natural and human-related ecosystems to unravel the population genomics and genome fluidity of bacteria. This will shed light on the species concept, the structure of bacterial populations, its interaction with viruses and the environment, their adaptation and their evolution, as well as opening new avenues for treating infectious diseases. In addition, a web-based server will be created to study, annotate and analyze bacterial genomes, and new scripts and computer programs will be developed that will be of great use for the scientific community. It is anticipated that the group will serve as a nucleating agent that will permit Spanish Microbiology to remain in the mainstream of international Microbiology and to continue being a support to the future of Spanish Biotechnology and Biomedicine.

The presence and role of low abundance microorganisms could explain the huge microbial diversity of natural systems. A case study in the Doñana National Park. Ministry of Science and Innovation, project CGL2009-12328/BOS.

Microbial diversity in natural environments is huge and difficult to determine. These microbial communities can be considered as formed by a low number of highly abundant microorganisms and a very high number of rare, low abundant, microorganisms. We present the hypothesis that the microorganisms representing that minority are important in natural ecosystems. The study of the rare microorganisms is essential for understanding the functional role of microbial communities in an spatial and temporal environment, as well as to comprehend why exist a so large microbial diversity. The selection of microorganisms adapted to thrive under extreme conditions (high temperature, low or high pH) from moderate environments is proposed. The selected environment is the sediment from the natural ponds at Doñana National Park. Environmental variables will be determined and changes in the microbial communities will be monitored during the selecting process. Molecular methods will be used for the detection of microorganisms based on both fingerprinting techniques and sequencing, and in situ detection methods. Cultures of selected microorganisms will be approached. Microorganisms, Bacteria and Archaea, will be identified and their physiological properties evaluated. Also, their spatial distribution will be analyzed and will contribute to decipher the potential function within their ecosystem. The possibility that those extreme conditions could represent natural situations will be studied. The analyzed processes will represent a model of the dynamic of microbial communities as a consecuence of environmental changes and their potential response both in the ecosystem and global biogeochemical cycles.

Microbial diversity and microbiology of extreme environments. Andalusian Government, BIO288.

Microbial diversity is a broad term that can be approached from very different perspectives. This includes not only the phylogenetic diversity but also includes molecular, functional, and mechanistics points of view. To achieve this broad assessment of the whole microbial world we use a wide and radically different methodologies which point towards a common objective, decipher the functioning of the microbial world. The focus on extremophiles allows to center on a specific type of microorganisms and communities which are of great interest for their potential biotechnological applications as well as model systesms for the study of natural communities.

IRNAS-CSIC, Avda. Reina Mercedes 10, 41012-Sevilla, Spain
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