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Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future.

During this century, a number of reports have described the potential roles of thermophiles in the upper soil layers during high-temperature periods. This study evaluates the capabilities of these microorganisms and proposes some potential consequences and risks associated with the activity of soil thermophiles. They are active in organic matter mineralization, releasing inorganic nutrients (C, S, N, P) that otherwise remain trapped in the organic complexity of soil. To process complex organic compounds in soils, these thermophiles require extracellular enzymes to break down large polymers into simple compounds, which can be incorporated into the cells and processed. Soil thermophiles are able to adapt their extracellular enzyme activities to environmental conditions. These enzymes can present optimum activity under high temperatures and reduced water content. Consequently, these microorganisms have been shown to actively process and decompose substances (including pollutants) under extreme conditions (i.e., desiccation and heat) in soils. While nutrient cycling is a highly beneficial process to maintain soil service quality, progressive warming can lead to excessive activity of soil thermophiles and their extracellular enzymes. If this activity is too high, it may lead to reduction in soil organic matter, nutrient impoverishment and to an increased risk of aridity. This is a clear example of a potential effect of future predicted climate warming directly caused by soil microorganisms with major consequences for our understanding of ecosystem functioning, soil health and the risk of soil aridity.

Microorganisms rule the functioning of our planet and each one of the individual macroscopic living creature. Nevertheless, microbial activity and growth status have always been challenging tasks to determine both in situ and in vivo. Microbial activity is generally related to growth, and the growth rate is a result of the availability of nutrients under adequate or adverse conditions faced by microbial cells in a changing environment. Most studies on microorganisms have been carried out under optimum or near-optimum growth conditions, but scarce information is available about microorganisms at slow-growing states (i.e., near-zero growth and maintenance metabolism). This study aims to better understand microorganisms under growth-limiting conditions. This is expected to provide new perspectives on the functions and relevance of the microbial world. This is because (i) microorganisms in nature frequently face conditions of severe growth limitation, (ii) microorganisms activate singular pathways (mostly genes remaining to be functionally annotated), resulting in a broad range of secondary metabolites, and (iii) the response of microorganisms to slow-growth conditions remains to be understood, including persistence strategies, gene expression, and cell differentiation both within clonal populations and due to the complexity of the environment.

Extremophiles possess unique cellular and molecular mechanisms to assist, tolerate, and sustain their lives in extreme habitats. These habitats are dominated by one or more extreme physical or chemical parameters that shape existing microbial communities and their cellular and genomic features. The diversity of extremophiles reflects a long list of adaptations over millions of years. Growing research on extremophiles has considerably uncovered and increased our understanding of life and its limits on our planet. Many extremophiles have been greatly explored for their application in various industrial processes. In this review, we focused on the characteristics that microorganisms have acquired to optimally thrive in extreme environments. We have discussed cellular and molecular mechanisms involved in stability at respective extreme conditions like thermophiles, psychrophiles, acidophiles, barophiles, etc., which highlight evolutionary aspects and the significance of extremophiles for the benefit of mankind

Today, it is definitively accepted that microorganisms play a central role in the functioning and maintenance of our planet and the organisms thriving on it. Nevertheless, we are starting to understand both how microorganisms function and the huge microbial diversity existing on Earth. As a result, we are beginning to comprehend and apply the implications of complex microbial communities and each of their specific members in both local (i.e., local ecosystems and/or organisms) and global (i.e., whole-planet change) processes. All animals and plants thriving on Earth depend on microbial activity for their growth and maintenance. Microbial speciation and persistence are topics that are yet to be well understood. Furthermore, microbial activity and the growth of microorganisms over a broad spectrum of environmental conditions are poorly understood, and therefore, our comprehension of microbial relevance, applications, and potential management issues await further advances in microbiology.

Chitinases are cosmopolitan lytic enzymes secreted by microbiomes that fall under the domain of fungi, yeasts, bacteria and plants. Most fungal plant pathogens, human infectious agents and post-harvest damage caused by pathogens have been a serious threat to the economy and human health. Like fungi, crabs, insects, lobsters, shrimps, and invertebrates all have a hard disintegrating, flexible polymer called chitin that forms the exterior skeleton. It poses a wide-range of environmental problem and a major threat to humans, plants and animals. According to functional genomic research, there is a large diversity of chitinases-producing fungi in nature. They have adapted to a wide range of habitats on Earth including plants, animals and manmade natural and artificial habitats. Chitinases, both native and genetically modified, have been produced and expressed in an expression system such as Escherichia coli or Pichia pastoris through recombinant DNA technology. This versatile recombinant chitinases can be used for long-term growth and productivity. As a whole, chitinases have a wide range of applications in agriculture, horticulture , plant health, and bio-control of pests, and even some fuel processing, genetically engineered molecule-based therapies, polysaccharide hydrolysis, biomedicine, pathogenic/virulence agents, antifungal agents, and as a drug delivery system.

Actinobacteria have been known to the world since the mid-eighteenth century, when Ferdinand Cohn portrayed them as ropy critters. In the nineteenth century, these unidentified morphons became famous for their broad array of applications and for causing maladies. Over the last five decades, actinomycetes have been classified and their names have been validly published as representative taxa. Actinobacteria have preserved their overall genetic integrity and kept newer genes that code for bioactive substances, as evidenced by their capacity to produce a myriad of bioactive compounds. As of today, we have hardly cultured 1% of existing microorganisms including Actinobacteria. Some Streptomyces strains were exploited for enhanced production of new bioactive compounds. In times of the bioinformatics era, insufficient and developing laboratory methods for enumeration of potential species are acting as a barrier to obtaining the maximum product in downstream processing. This issue may be addressed by using the reserve-engineering approach. Published reports indicate that new or high production can be achieved through either strain improvement or the remaining microbial dark matter consists of growing hidden gems for an awaited potential application. Reverse engineering of microorganisms, on the other hand, improves the efficacy of a specific target gene to obtain high product value, which can be seen in the expression of the tylosin biosynthetic gene cluster. Hidden gems among the phylum Actinobacteria need to be discovered and reverse engineered for antibiotics and novel drugs. Therefore, advancement in research machine learning and artificial intelligence seems to be incredible tools. Hence, uncultivated Actinobacteria can be used for the benefit of mankind by combining reverse engineering with machine learning (ML) and artificial intelligence (AI).

Figure 2. Molecular tunnel dimensions, length (A, D, G), surface (B, E, H) and volume (C, F, I) (in Å), predicted for the methyl-CoM reductase (Mmr) gene subunits, alpha (A, B, C), beta (D, E, F) and gamma (G, H, I), from numerous methanogenic Archaea as a function of their optimum growth rates. Dark color filled symbols correspond to predicted protein structures. Light color filled symbols correspond to resolved model structures from the Protein Data Bank.

Figure 4. Amplitude and average of the molecular tunnel dimensions, length (A, D, G), surface (B, E, H) and volume (C, F, I) (in Å), predicted for the methyl-CoM reductase (Mmr) gene subunits, alpha (A, B, C), beta (D, E, F) and gamma (G, H, I) from methanogenic Archaea classified in three classes of optimum growth temperature (<50ºC; 50-80ºC; >80ºC). Grey bars correspond to the amplitude of data observed for estimates of the molecular tunnel dimensions. Red squares represent the average dimensions for each Figure 5. Amplitude and average of the molecular tunnel dimensions, length (A, D, G), surface (B, E, H) and volume (C, F, I) (in Å), predicted for the heterodisulfide reductas (Hdr) gene subunits, A (A, B, C), B (D, E, F) and C (G, H, I) from methanogenic Archaea classified in three classes of optimum growth temperature (<50ºC; 50-80ºC; >80ºC). Grey bars correspond to the amplitude of data observed for estimates of the molecular tunnel dimensions. Red squares represent the average dimensions for each growth temperature class. Error bars for the average values represent a standard deviation.

Analyses of protein structures have shown the existence of molecular channels in enzymes from Prokaryotes. Those molecular channels suggest a critical role of spatial voids in proteins, above all, in those enzymes functioning under high temperature. It is expected that these spaces within the protein structure are required to access the active site and to maximize availability and thermal stability of their substrates and cofactors. Interestingly, numerous substrates and cofactors have been reported to be highly temperature-sensitive biomolecules. Methanogens represent a singular phylogenetic group of Archaea that performs anaerobic respiration producing methane during growth. Methanogens inhabit a variety of environments including the full range of temperatures for the known living forms. Herein, we carry out a dimensional analysis of molecular tunnels in key enzymes of the methanogenic pathway from methanogenic Archaea growing optimally over a broad temperature range. We aim to determine whether the dimensions of the molecular tunnels are critical for those enzymes from thermophiles. Results showed that at increasing growth temperature the dimensions of molecular tunnels in the enzymes methyl-coenzyme M reductase and heterodisulfide reductase become increasingly restrictive and present strict limits at the highest growth temperatures, i.e., for hyperthermophilic methanogens. However, growth at lower temperature allows a wide dimensional range for the molecular spaces in these enzymes. This is in agreement with previous suggestions on a potential major role of molecular tunnels to maintain biomolecule stability and activity of some enzymes in microorganisms growing at high temperatures. These results contribute to better understand archaeal growth at high temperatures. Furthermore, an optimization of the dimensions of molecular tunnels would represent an important adaptation required to maintain the activity of key enzymes of the methanogenic pathway for those meth

El objetivo general del proyecto es dar a conocer la Ciencia y la actividad investigadora del Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), a través de la frescura de las ilustraciones de dibujantes aficionados y Urban Sketcher, que aportan un carácter informal y divertido pero riguroso, mediante el poder del ideograma: una imagen vale más que mil palabras. Este proyecto fomenta la sinergia que existe entre la ciencia y el arte, para darle a la investigación que se realiza en el IRNAS una dimensión estética y simplificadora que favorece la divulgación. Se pretende así incrementar la cultura científica, tecnológica e innovadora de la sociedad y facilitar su acceso a los resultados científicos. A todos los dibujantes que han participado se les ha invitado a visitar los laboratorios e invernaderos del Instituto y la finca experimental, donde todas y todos los implicados, les han explicado sus proyectos, resultados y aplicaciones. Con esta información y sus observaciones, han realizado los dibujos que se muestran a continuación y que reflejan aspectos de la investigación realizada en el IRNAS. Este libro es el resultado final del proyecto, tanto en versión física como digital, y en él se recogen los dibujos realizados a lo largo de tres meses (octubre-diciembre 2022). Más información del proyecto en www.irnas.csic.es. La participación multidisciplinar en este proyecto ha sido vital para obtener diferentes puntos de vista, ya que los dibujantes provienen de muy diversos campos (ingeniería en edificación, arquitectura, bellas artes, magisterio, dibujo publicitario, biología, medicina, informática, etc.), y cada uno aporta su visión de los trabajos de investigación, enriqueciendo así el resultado. Esperamos que esta iniciativa sirva para impulsar la interrelación arte-ciencia, contribuyendo a una sociedad más informada y receptiva al conocimiento científico

Los suelos son ambientes muy heterogéneos que albergan una diversidad microbiana extremadamente elevada. Además, los microorganismos desempeñan un papel decisivo en los ciclos biogeoquímicos de los elementos contribuyendo al mantenimiento y buen funcionamiento del ecosistema suelo. Sin embargo, nuestro conocimiento sobre el funcionamiento de los microorganismos del suelo y sus consecuencias en relación al cambio climático aún es limitado. Este estudio se centrará en la presencia, funcionamiento y consecuencias de los microorganismos termófilos presentes en suelos como un modelo de estudio de un tipo de microorganismos generalmente discriminado en las comunidades microbianas del suelo. Los termófilos del suelo están mayoritariamente representados por Firmicutes, principalmente relacionados con el género (Para)Geobacillus. Estas bacterias se encuentran mayoritariamente como células viables y participan activamente en los ciclos del C, N, S y P procesando la materia orgánica del suelo y liberando nutrientes que quedan disponibles a plantas y otros microorganismos. El paso limitante para la descomposición de los compuestos orgánicos complejos del suelo son las enzimas extracelulares de los microorganismos. Las enzimas extracelulares de los microorganismos termófilos del suelo además de funcionar óptimamente a temperaturas elevadas parecen adaptarse a a condiciones de bajo contenido hídrico. Estas condiciones son típicas en las capas superiores de suelos durante períodos cálidos y de sequía. Como consecuencia del cambio climático, estos eventos de desecación y elevada temperatura se prevé que aumenten en frecuencia y duración. Por tanto, la actividad de estos termófilos se espera que aumente en el futuro. El papel de estos termófilos es de interés para comprender el funcionamiento de suelos, especialmente suelos áridos, y sus posibles consecuencias a distintas escalas, tanto a nivel local en el ecosistema como a nivel global.

Thermophiles thrive under high temperatures unlike other organisms. In order to grow under those high temperature conditions, the biomolecules supporting life of thermophiles must remain stable under those conditions. The relationship of temperature to the structure of macromolecules (e.g., proteins, nucleic acids) has been studied for many years. Nevertheless there are some additional points that remain to be deciphered in order to explain how these cells are able to live at high temperatures. This study will focus on clarifying some of these aspects in order to better understand how life is possible at high temperatures. For example, a number of small biomolecules (e.g., NADH, ATP, pyridoxal phosphate, etc.) are highly sensitive to high temperatures but they are basic metabolic constituents for all organisms. We have shown that NADH, as an example, can be stabilized if cellular viscosity is maintained and most cells (mesophiles and thermophiles) are able to do so. Additional mechanisms might be required at the highest temperatures for life (i.e., >80ºC). A mechanisms that has been suggested are the molecular channels in the enzymes which will assist in interactions between substrates, cofactors and the enzymes. Interestingly the dimensions of the molecular channels are increasingly restrictive at increasing growth temperatures. Increasing growth temperatures are expected to increase molecular mobility and interactions and so decrease stability of biomolecules at high temperatures. Measuring fluorescence lifetime of proteins in vivo, it is being tested that thermophiles can maintain the mobility of amino acid residues. This suggests that the intracellular environment in hyperthermophilic cells is highly regulated and adapted to thrive at the highest temperatures supporting the growth of hyperthermophiles. Thermophiles require high temperature to grow and the above results contribute to better understand how this is possible.

The Acidobacteria phylum has been frequently detected in a variety of environments but it remains scarcely studied, in part, because it only has a few representative cultured species. One of the cultured Acidobacteria is the species Acidobacterium capsulatum. A. capsulatum is an acidophilic, aerobic, heterotrophic bacterium which grows optimally at pH 3.5 and 30ºC. Whereas most bacteria have been studied under optimum, or near optimum, conditions, scarce information is available on their capability and physiology under growth-limiting conditions and even on the possibility of obtaining these cells growing at very low rates. Herein, we study the properties of A. capsulatum over a range of growth rates focusing on low rates. At each growth rate, we analyzed several parameters such as the morphology, culturability, fluorescent determination of live/dead counts, cell membrane potential and estimates of RNA/DNA ratio. The relationships among these variables are studied to realize the growing properties of these cells under drastically different growth rates. The capacity of A. capsulatum to persist in nature through severe growth-limiting conditions is evaluated in base to our results.

La genómica es una ciencia que nos permite comprender la relación entre la información genética codificada en el ADN y la fisiología del microorganismo, así como aspectos bioquímicos y evolutivos. Se secuenció y ensambló el genoma de Parageobacillus thermoglucosidasius 23.6, una cepa termófila aislada del suelo en el Parque Nacional de Doñana (Huelva) (CECT9776). La secuenciación identificó 4 moléculas de ADN circular: El genoma principal o Tmp1 (3.870.682 pb), Tmp2 (102283 bp), un megaplásmido, Tmp3 (50843 pb), un plásmido, y un bacteriófago, Tmp4 (55505 pb). El pangenoma de la cepas de P. thermoglucosidaius es abierto y ha identificado en Tmp1 y Tmp2 puntos calientes de transferencia horizontal de genes constituido por genes únicos y elementos genéticos móviles (transposasas). El sesgo del uso de codones establece una relación entre los genes únicos y Tmp3, que contiene en su secuencia un elemento integrativo y conjugativo. El bacteriófago identificado pertenece a la familia Syphoviridae y no está integrado en el genoma principal. El gran número de regiones CRISPR presentes en Tmp1 y la homología de 11 espaciadores con Tmp4 podría explicar porque el fago no se integra en el genoma principal. La información genómica de Parageobacillus thermoglucosidasius 23.6 sugiere que podría tener la plasticidad de incorporar ADN a su genoma a través de diferentes estrategias de transferencia horizontal de genes (transformación, conjugación y traducción), lo que facilitaría su capacidad de adaptación a nuevos medios y a cambios producidos en el ambiente.

oils are highly heterogeneous and support highly diverse microbial communities. Microbial extracellular enzymes breakdown complex polymers into small assimilable molecules representing the limiting step of soil organic matter mineralization. This process occurs on to soil particles although currently it is typically estimated in laboratory aqueous solutions. Herein, estimates of microbial extracellular enzyme activity were obtained over a broad range of temperatures and water availabilities frequently observed at soil upper layers. A Pseudomonas strain presented optimum extracellular enzyme activities at high water activity whereas a desiccation resistant bacterium (Deinococcus) and a soil thermophilic isolate (Parageobacillus) showed optimum extracellular enzyme activity under dried (i.e., water activities ranging 0.5-0.8) rather that wet conditions. Different unamended soils presented a distinctive response of extracellular enzyme activity as a function of temperature and water availability. This study presents a procedure to obtain realistic estimates of microbial extracellular enzyme activity under natural soil conditions of extreme water availability and temperature. Improving estimates of microbial extracellular enzyme activity contribute to better understand the role of microorganisms in soils.

On 20th July 1969, the Fresnedillas Control Station, near Madrid, received the first words of a human from the surface of the Moon. “That’s one small step for [a] man, one giant leap for mankind”, was the historical sentence recorded from Neil A. Armstrong, commander of the “Apollo XI” mission. Nowadays, fifty years after Armstrong’s epic achievement, space exploration by humans is commonly recognized as a highly exciting and attractive challenge and a powerful booster for scientific and technological progress in order to improve the human life on Earth (NASA et al. 2018). This is true despite some criticisms (minority, but significant) that question the high costs that it entails (Rinaldi 2016). The establishment of permanent settlements in the Moon and Mars is becoming a realistic venture day by day. After a decade of successful rover explorations to the surface of Mars (Voosen 2018), both ESA and NASA, and more recently the agencies from growing economies in Asian countries, are working to promote a manned mission, first to the Moon, and then to Mars. The European Space Agency (ESA), of which Spain is an active member, adheres to these objectives and is strongly committed in supporting and participating in these programs. The main aim of space life science is to understand how the space environment, and specifically altered gravity and radiation, affects the morphology, physiology and behaviour of living organisms, and to design countermeasures to enable terrestrial life, and particularly human life, to develop outside Earth. That is, how they perceive and respond to gravity and radiation and adapt to the space environment. There is a variety of disciplines, such as genetics, molecular, anatomical or physiological fields, which use a range of technologies to address these issues. In order to understand adaptations at the functional level it is necessary to comprehend adaptations at cellular and tissue levels. Also, basic research analysing biomolecules, cells and

Evolutionary biology seeks to understand how biological diversity originates and is maintained. High-throughput sequencing permits assembling chromosome-level genomes, characterizing single-cell transcriptomes, and determining epigenomic modifications. Once widely applied to the diversity of living organisms, the reconstruction of the Tree of Life and the identification of the genomic targets of natural selection will be achieved

Sulfur (S) is a macro-element required for life. S deficiency limits plant growth. Microorganisms carry out several essential steps in the recycling of organic and inorganic S in soils. Microbes and plants interact, mainly in the rhizosphere, but the mechanisms ruling these interactions and the extent of such relationships remain poorly understood. Here, we update current perspectives on the role of specific microorganisms involved in S cycling and the spatial interaction between plants and microbes. To contextualize the pitfalls of current approaches in studying soil S transformations, we review the current main established steps, redox reactions and microbial players in the S cycle. The incorporation of novel microbial taxa, namely those important for organic S mineralization, which may be important ecosystem players in terms of soil functionality, and of the spatial-temporal context at aggregate-level for the relevance of plant-microbe interactions, introduce important implications involving the role of microorganisms in the rhizosphere and require an integrated analysis. Herein, the rhizosphere is a focus – a habitat of selected low-abundance species, where important microbial groups act in S turnover and plant growth – while keeping a perspective on important microbial feedback S fluxes that may occur in bulk soils.

This study aims to determine the diversity of culturable thermophilic bacteria isolated from eight terrestrial hot springs in Northeastern of Algeria using the conventional methods, SDS-PAGE fingerprinting of whole-cell proteins and 16S rRNA gene sequencing. In addition, their hydrolytic enzyme activities were also investigated. A total of 293 strains were isolated from the hot springs’ water and sediment using different culture media. Overall, five distinct bacterial groups were characterized by whole-cell protein pattern analysis. Based on the 16S rRNA gene sequencing of 100 selected strains, the isolates were assigned to the following three major phyla: Firmicutes (93%), Deinococcus-Thermus (5%), and Actinobacteria (2%), which included 27 distinct species belonging to 12 different phylotypes, Aeribacillus, Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Laceyella, Meiothermus, Saccharomonospora, Thermoactinomyces, Thermobifida, and Thermus. The screening for nine extracellular enzymes showed that 65.87% of the isolates presented at least five types of enzyme activities, and 6.48% of strains combined all tested enzymes (amylase, cellulase, pectinase, esculinase, protease, gelatinase, lipase, lecithinase, and nuclease). It was found that Bacillus, Anoxybacillus, Aeribacillus, and Aneurinibacillus were the genera showing the highest activities. Likewise, the study showed an abundant and diverse thermophilic community with novel taxa presenting a promising source of thermozymes with important biotechnological applications. This study showed that a combined identification method using SDS-PAGE profiles of whole-cell proteins and subsequent 16S rRNA gene sequence analysis could successfully differentiate thermophilic bacteria from Algerian hot springs.

The taxonomic concept of species has received continuous attention. A microbial species as a discrete box contains a limited number of highly similar microorganisms assigned to that taxon, following a polyphasic approach. In the 21st Century, with the advancements of sequencing technologies and genomics, the existence of a huge prokaryotic diversity has become well known. At present, the prokaryotic species might no longer have to be understood as discrete values (such as 1 or 2, by homology to Natural numbers); rather, it is expected that some microorganisms could be potentially distributed (according to their genome features and phenotypes) in between others (such as decimal numbers between 1 and 2; real numbers). We propose a continuous species concept for microorganisms, which adapts to the current knowledge on the huge diversity, variability and heterogeneity existing among bacteria and archaea. Likely, this concept could be extended to eukaryotic microorganisms. The continuous species concept considers a species to be delimited by the distance between a range of variable features following a Gaussian-type distribution around a reference organism (i.e., its type strain). Some potential pros and cons of a continuous concept are commented on, offering novel perspectives on our understanding of the highly diversified prokaryotic world, thus promoting discussion and further investigation in the field. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

In Portugal, many abandoned mines are often close to agricultural areas and might be used for plant food cultivation. Soils in the vicinity of two Mn- and Fe-abandoned mines (Ferragudo and Rosalgar, SW of Portugal) were collected to cultivate two different food species (Brassica rapa subsp. pekinensis (Lour.) Hanelt and Origanum vulgare L.). Chemical characterization of the soil–plant system and potential risk of adverse effects for human health posed by plants associated with soil contamination, based on the estimation of hazard quotient (HQ), were assessed in a microcosm assay under greenhouse conditions. In both soils, the average total concentrations of Fe and Mn were above the normal values for soils in the region and their concentration in shoots of both species was very high. Brassica rapa subsp. pekinensis grew better in Ferragudo than in Rosalgar soils, and it behaved as an excluder of Cu, Mn, Fe, S and Zn in both soils. The HQ for Cu, Fe, Mn and Zn in the studied species grown on both soils was lower than unit indicating that its consumption is safe. The high Mn tolerance found in both species might be due in part to the high contents of Fe in the soil available fraction that might contribute to an antagonism effect in the uptake and translocation of Mn. The obtained results emphasize the need of further studies with different food crops before cultivation in the studied soils to assess health risks associated with high metal intake.

The effect of exposure to high Mn concentration was studied in a metallophyte species, Erica andevalensis, using hydroponic cultures with a range of Mn concentrations (0.06, 100, 300, 500, and 700 mg L−1). At harvest, biomass production, element uptake, and biochemical indicators of metal stress (leaf pigments, organic acids, amino acids, phenols, and activities of catalase, peroxidase, superoxide dismutase) were determined in leaves and roots. Increasing Mn concentrations led to a decrease in biomass accumulation, and tip leaves chlorosis was the only toxicity symptom detected. In a similar way, photosynthetic pigments (chlorophylls a and b, and carotenoids) were affected by high Mn levels. Among organic acids, malate and oxalate contents in roots showed a significant increase at the highest Mn concentration, while in leaves, Mn led to an increasing trend in citrate and malate contents. An increase of Mn also induced an increase in superoxide dismutase activity in roots and catalase activity in leaves. As well, significant changes in free amino acids were induced by Mn concentrations higher than 300 mg L−1, especially in roots. No significant changes in phenolic compounds were observed in the leaves, but root phenolics were significantly increased by increasing Mn concentrations in treatments. When Fe supply was increased 10 and 20 times (7–14 mg Fe L−1 as Fe-EDDHA) in the nutrient solutions at the highest Mn concentration (700 mg Mn L−1), it led to significant increases in photosynthetic pigments and biomass accumulation. Manganese was mostly accumulated in the roots, and the species was essentially a Mn excluder. However, considering the high leaf Mn concentration recorded without toxicity symptoms, E. andevalensis might be rated as a Mn-tolerant species.

The biostimulant potential of three different organic acids (OAs) present in the rhizosphere, specifically lactic, oxalic, and citric acids, have been studied. The results showed a rapid and complete metabolism of these three acids with soil microorganisms using them as a source of carbon and energy. Biostimulation was confirmed by soil biochemical studies which showed an increase in enzymatic activities, such as dehydrogenase and phosphatase, lactic and citric acids being those that produced the greatest biostimulation. With regard to microbiota composition, amplicon sequencing of the 16S rRNA gene showed changes in the structure of soil microbial communities. Applying OAs produced a decrease in richness and diversity indices, inducing specific changes in the structure of the microbiological communities. Applying lactic acid induced rapid changes in microbiota composition at both phylum and family taxonomic levels, favoring the proliferation of microorganisms involved in its degradation and soil fertility, such as the genus Bacillus and the family Micrococcaceae. Once the lactic acid was degraded, the biodiversity tended to return to similar phyla, but specific distinctive families and genera remained, leaving a pattern of induction of taxa described as plant growth-promoting bacteria (PGPB), such as the Sinorhizobium and Lysobacter genera, and the Pseudomonaceae family. Similar behavior was found with citric acid, which favored the proliferation and dominance of microorganisms of the Clostridiaceae family, involved in its degradation, as well as microorganisms of both the Micrococcaceae and Pseudomonadaceae families which were found on day 7, leaving a similar pattern of induction as that found after the mineralization of lactic acid. On the other hand, oxalic acid induced long-lasting changes in the bacterial community composition. This was characterized by an increase in the proportion of the Burkholderiales order, which includes microorganisms involved in the d

Figure 2. Decay rates as a function of water availability and temperature for extracellular enzymes from mesophiles and thermophiles at three different soils. Extracellular enzymes: A, B and C (upper row), glucosidases; D, E and F (central row), phosphatases; G, H and I (lower row), proteases. Left column (A, D and G), Seville soil (South Spain); Center column (B, E and H), Cadiz soil (South Spain); Right column (C, F and I), North Spain soil. Symbols: in red, decay of extracellular enzymes from thermophiles at 60ºC; in blue, decay of extracellular enzymes from mesophiles at 20ºC; in black, decay of extracellular enzymes from thermophiles at 20ºC. Points resulted from the average of triplicated samples. Error bars indicate a standard deviation, Figure 3. NMDS ordination of decay rates as a function of temperature, water availability and the soils for glucosidase (A), phosphatase (B) and protease (C) activities. Water activity is shown with brownish filled circles from dark to light in decreasing levels of water activity. Decays at 20ºC and 60ºC by enzymes from mesophilic (M20, down-pointing triangles, and M60, up-pointing triangles, respectively) and thermophilic (T20, squares, and T60, diamonds, respectively) microoorganisms are shown as unfilled symbols for each studied soil (Huesca soil [North Spain] in green, Cadiz soil [South Spain] in blue, Seville soil [South Spain] in red).

Microbial extracellular enzyme activity (EEA) is critical for the decomposition of organic matter in soils. Generally, EEA represents the limiting step governing soil organic matter mineralization. The high complexity of soil microbial communities and the heterogeneity of soils suggest potentially complex interactions between microorganisms (and their extracellular enzymes), organic matter and physicochemical factors. Previous studies have reported the existence of maximum soil EEA at high temperatures although microorganisms thriving at high temperature represent a minority of soil microbial communities. To solve this paradox, we attempt to evaluate if soil extracellular enzymes from thermophiles could accumulate in soils. Methodology at this respect is scarce and an adapted protocol is proposed. Herein, the approach is to analyze the persistence of soil microbial extracellular enzymes at different temperatures and under a broad range of water availability. Results suggest that soil high temperature EEA presented longer persistence than enzymes with optimum activity at moderate temperature. Water availability influenced enzyme persistence, generally preserving for longer time the extracellular enzymes. These results suggests that hightemperature extracellular enzymes could be naturally accumulated in soils. Thus, soils could contain a reservoir of enzymes allowing a quick response by soil microorganisms to changing conditions. This study suggests the existence of novel mechanisms of interaction among microorganisms, their enzymes and the soil environment with relevance at local and global levels.

Examples of the decay curves for extracellular enzymes from mesophiles and thermophiles at 20ºC and 60ºC in a South Spain (Seville) soil under water activity 1 (wet conditions). Red circles, decay of enzyme activity from thermophiles at 60ºC; pink circles, decay of enzyme activity from thermophiles at 20ºC; black triangles, decay of enzyme activity from mesophiles at 20ºC; grey triangles, decay of enzyme activity from mesophiles at 60ºC. Points are average values from triplicates. Error bars indicate a standard deviation

Scattered plots showing the relationship between water activity (X-axis), percentage of maximum enzyme activity estimates at 20ºC (left) and 60ºC (right) (proportional to diameter of circles) with environmental variables (Y-axis), specifically, two climaterelated parameters, the annual average of hot days (>30ºC)(A) and the annual average of consecutive days without precipitation (B), and soil-texture through the fraction of sand in the sampled soils (C). Symbol colors indicate the type of enzyme (Dark to light: Glucosidase, Phosphatase, Protease) and the analyzed soil (Greenish, Galicia (G); bluish, Aragón (P); brownish, Salamanca (S); reddish, Sevilla (C); purplish, Cádiz (T)).

RDA plot showing the correspondence of water activity giving the optimum enzyme activity and environmental parameters. Capital letters (in black) represent the sampled soils (G, Galicia, P, Aragón; S, Salamanca; C, Sevilla; T, Cádiz). Arrows represent the environmental variables (soil texture, sand and silt content) contributing significantly to explain the variability of water activity resulting in optimum enzyme activity. The distribution of enzyme activities are presented in red: Glu_20, glucosidase activity at 20ºC; Glu_60, glucosidase activity at 60ºC; Pho_20, phosphatase activity at 20ºC; Pho_60, phosphatase activity at 60ºC; Pro_20, protease activity at 20ºC; Pro_60, protease activity at 60ºC. Figure

Microorganisms govern soil carbon cycling with critical effects at local and global scales. The activity of microbial extracellular enzymes is generally the limiting step for soil organic matter mineralization. Nevertheless, the influence of soil characteristics and climate parameters on microbial extracellular enzyme activity (EEA) performance at different water availabilities and temperatures remains to be detailed. Different soils from the Iberian Peninsula presenting distinctive climatic scenarios were sampled for these analyses. Results showed that microbial EEA in the mesophilic temperature range present optimal rates under wet conditions (high water availability) while activity at the thermophilic temperature range (60ºC) could present maximum EEA rates under dry conditions if the soil is frequently exposed to high temperatures. Optimum water availability conditions for maximum soil microbial EEA were influenced mainly by soil texture. Soil properties and climatic parameters are major environmental components ruling soil water availability and temperature which were decisive factors regulating soil microbial EEA. This study contributes decisively to the understanding of environmental factors on the microbial EEA in soils, specifically on the decisive influence of water availability and temperature on EEA. Unlike previous belief, optimum EEA in high temperature exposed soil upper layers can occur at low water availability (i.e., dryness) and high temperatures. This study shows the potential for a significant response by soil microbial EEA under conditions of high temperature and dryness due to a progressive environmental warming which will influence organic carbon decomposition at local and global scenarios.

This paper presents a simple, swift, improved and inexpensive one plate-double nutrient endospore activation method (ODNEAM) for activation of thermophilic endospore (thermospore). Revival and cultivation of dormant bacteria experiencing un-culturable state is a difficult task. Long-term preservation at low temperature (4 °C or below) under laboratory conditions pushes most of the gram-positive bacteria to form endospores and enter dormancy. After experienced dormancy and crossed specific time (more than a month) it’s difficult for those thermospores to resurrect into a living cell. The effect of low temperature, a double quantity of nutrients and high humidity (80 %) was re-revaluated to foster germination of thermospores. Employed parameters successfully revived thermospores which experience dormancy or unable to initiate the germination process upon onset of favorable conditions. This method has crucial importance for activation of the dormant thermospore. Long-term preservation under low temperature or no sub-culturing at a fixed time interval of thermophilic bacteria may be a cause for least revival rate and the progressively diminished revival rate. The inactivation of cells after long-term preservation and probable loss to science would be avoided using this one plate-double nutrient endospore activation method. This is a tribute to Harold F. Foerster−A renowned microbiologist (1983).

Figure 2. Extracellular glucosidase activity at 20ºC (A) and 60ºC (B) as a function of water activity for different soils. Enzyme activity is presented as percentage of maximum activity for each soil. Error bars represent the standard deviation. Symbols represent different soils: Black square, Galicia; Grey square, Aragón; Black triangle, Salamanca; Grey triangle, Seville; Black circle and dashed line, Cádiz. Figure 3. Extracellular phosphatase activity at 20ºC (A) and 60ºC (B) as a function of water activity for different soils. Enzyme activity is presented as percentage of maximum activity for each soil. Error bars represent the standard deviation. Symbols represent different soils: Black square, Galicia; Grey square, Aragón; Black triangle, Salamanca; Grey triangle, Seville; Black circle and dashed line, Cádiz. Figure 4. Extracellular protease activity at 20ºC (A) and 60ºC (B) as a function of water activity for different soils. Enzyme activity is presented as percentage of maximum activity for each soil. Error bars represent the standard deviation. Symbols represent different soils: Black square, Galicia; Grey square, Aragón; Black triangle, Salamanca; Grey triangle, Seville; Black circle and dashed line, Cádiz.

El uso excesivo de plásticos tales como el polipropileno representa un serio problema ambiental agravado con la llegada de la pandemia y el desecho constante de mascarillas higiénicas, fabricadas con fibras de este material. En este trabajo planteamos el estudio de la degradación microbiana de mascarillas debido a la larga persistencia del polipropileno en la naturaleza. Realizamos cultivos de enriquecimiento de microorganismos procedentes de una muestra natural de suelo suplementados con fragmentos de mascarillas. La identificación de los microorganismos que componen el enriquecimiento obtenido se llevó a cabo por secuenciación de su metagenoma (shotgun sequencing). Con objeto de simular posibles efectos físico-químicos sobre la biodegradabilidad del material, se analizaron una serie de factores, incluyendo pre-tratamientos térmicos, enzimáticos, con metales y la presencia de surfactantes. Las fibras se evaluaron mediante análisis microscópico. El enriquecimiento obtenido mostraba un aparente inicio de descomposición en anaerobiosis. Los componentes mayoritarios de la comunidad microbiana enriquecida fueron Franconibacter daqui, Desulfotomaculum aeronauticum, Sedimentibacter sp. y Clostridium intestinale. Cultivos de ese enriquecimiento en medio destinado al crecimiento de bacterias reductoras de sulfato dió lugar a la presencia de partículas de sulfuro metálico adheridas a la mascarilla lo que sugería que Desulfotomaculum, una típica bacteria reductora de sulfatos, podría estar implicada en la degradación de las fibras de las mascarillas. Los análisis microscópicos de fibras han permitido observar la asociación de estas bacterias a las fibras. Visualmente no se han observado efectos debido a los pretratamientos físico-químicos evaluados. Resultados previos obtenidos mediante RMN (Resonancia Magnética Nuclear) han demostrado que esta técnica permite caracterizar los productos resultantes de la rotura de las fibras de mascarillas incubadas en suelos [1]. En la actua

Halogenated compounds are environmental pollutants toxic to humans and wildlife. Certain microorganisms degrade these halogenated compounds. However, little is known about the potential of microorganisms in bioremediation under extreme conditions, specifically in arid and semi-arid soils frequently exposed to high temperatures and desiccation periods. Arid and semi-arid environments and deserts make up vast areas of Earth's landmass. To investigate the degradation of 2-chlorophenol (2-CP) in soils as a function of temperature and water availability, three bacterial species were tested, two soil mesophiles of the genus Rhodococcus, R. opacus and R. erythropolis, and a soil thermophilic isolate, Parageobacillus thermoglucosidasius. Degradation trials in soil samples with these species were performed over a range of water activity from 1 to 0.4. At their optimum growth temperature, R. opacus showed maximum 2-CP degradation at water activity 0.9 sharply decreasing when lowering water activity. Nevertheless, the Parageobacillus isolate (optimum growth temperature 60°C) showed maximum 2-CP degradation rates at water activity 0.5 which represented highly desiccating conditions. Parageobacillus degradation of 2-CP was very low at water activity above 0.9. Thus, biodegradation of 2-CP in soils is possible even under arid conditions although different microbial species might be involved in this task depending on the interactions of abiotic factors and the diversity of microbial communities in soils. These results contribute to understand the potential biodegradation of specific halogenated compounds in the environment which is of great relevance to comprehend the fate of halogenated pollutants (i.e., 2-CP) in deserts, arid and semi-arid soils.

The full biotechnological exploitation of thermostable enzymes in industrial processes is necessary for their commercial interest and industrious value. The heat-tolerant and heat-resistant enzymes are a key for efficient and cost-effective translation of substrates into useful products for commercial applications. The thermophilic, hyperthermophilic, and microorganisms adapted to extreme temperatures (i.e., low-temperature lovers or psychrophiles) are a rich source of thermostable enzymes with broad-ranging thermal properties, which have structural and functional stability to underpin a variety of technologies. These enzymes are under scrutiny for their great biotechnological potential. Temperature is one of the most critical parameters that shape microorganisms and their biomolecules for stability under harsh environmental conditions. This review describes in detail the sources of thermophiles and thermostable enzymes from prokaryotes and eukaryotes (microbial cell factories). Furthermore, the review critically examines perspectives to improve modern biocatalysts, its production and performance aiming to increase their value for biotechnology through higher standards, specificity, resistance, lowing costs, etc. These thermostable and thermally adapted extremophilic enzymes have been used in a wide range of industries that span all six enzyme classes. Thus, in particular, target of this review paper is to show the possibility of both high-value–low-volume (e.g., fine-chemical synthesis) and low-value–high-volume by-products (e.g., fuels) by minimizing changes to current industrial processes

El estudio de los microorganismos termófilos ha tenido un gran auge en las últimas décadas debido a la aplicación de sus enzimas y moléculas en procesos biotecnológicos. En la actualidad, existe un déficit en cuanto a comprender cómo viven los microorganismos en la naturaleza y sus estrategias de persistencia y adaptación. La presente tesis estudia la vida de un microorganismo termófilo aislado del suelo, Parageobacillus thermoglucosidasius 23.6, mas allá de la condiciones consideradas óptimas para su crecimiento. El desarrollo de bioreactores en continuo (quimiostato) con retención de células (retentostato), nos ha permitido cultivar la cepa 23.6 a tasas de crecimiento cercanas a cero (0,0002 h-1). Estas condiciones asumimos que son frecuentes en suelos, por la limitación de nutrientes, y por la influencia de ciclos diarios de temperatura (60 - 20º C). La secuenciación del genoma ha identificado 4 moléculas de ADN circular: El genoma principal o Tmp1, dos plásmidos, Tmp2 y Tmp3, y un bacteriófago, Tmp4, Su genoma ha servido de referencia para los análisis del transcriptoma. El pangenoma de la cepas de P. thermoglucosidaius es abierto y ha identificado puntos calientes de transferencia horizontal de genes llevados a cabo a través de diferentes estrategias: transformación, conjugación y traducción. El análisis del transcriptoma a tasas de crecimiento cercanas a cero reveló una gran sobreexpresión de genes relacionados con la obtención y búsqueda de energía, mecanismos de defensa y plasticidad genómica, permitiendo la adaptación de P. thermoglucosidasius 23.6 a nuevos ambientes y manteniendo su viabilidad. A bajas temperaturas (20º C) y tasa de crecimiento reducida (0.025h-1) hay una intensa respuesta transcripcional haciendo posible que la cepa 23.6 mantenga un metabolismo activo a bajas temperaturas que le permite responder rápidamente cuando las condiciones vuelvan eventualmente a ser óptimas para su crecimiento. Esta tesis contribuye a comprender el comportamiento

Índice Microorganismos Abundancia y diversidad Funciones y servicios Ciclos biogeoquímicos Diversidad ¿ Función ¿ Respuesta a cambios ambientales ¿ Salud del suelo Ejemplo Microorganismos termófilos del suelo Degradación contaminantes Participación en los ciclos del C, N, S, P Utilización de la materia orgánica Desecación y alta temperatura Reseñas y perspectivas