Putative ancient microorganisms from amber nuggets

Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganismlike specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified. [Int Microbiol 2007; 10(2):117-122

Isolation and taxonomic study of a new canthaxanthin-containing bacterium, Gordonia jacobaea MV-1 sp. nov

This article describes the isolation and taxonomic study of a coryneform isolate of a new Gordonia species (G. jacobaea), strain MV-1, which accumulates several carotenoids, including the ketocarotenoid trans-canthaxanthin. Identification of this new isolate by morphobiochemical methods did not allow unambiguous taxon assignment, but sequencing of the 16S rRNA gene clearly pointed to the genus Gordonia, Gordonia sputi being the closest fit. Differences in certain transversions/ transitions in otherwise very well-conserved sequences of the described Gordonia species supported the proposal of this new taxon. The fact that both the best growth and best pigmentation were obtained with glucose, an inexpensive carbon source and at an industrially suitable temperature, suggests that this new bacterial strain may have good potential for the industrial production of canthaxanthin

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production

Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e. β-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention. The bacterium Gordonia jacobaea readily synthesizes and accumulates large amounts of canthaxanthin (β-β´-carotene-4,4´-dione), which is widely used in the food and cosmetics industries. In the present work, the effects of different low-cost raw materials on fermentation and canthaxanthin accumulation by a hyperpigmented strain of G. jacobaea were studied. Canthaxanthin production and peak levels of accumulation varied according to the different media used. [Int Microbiol 2005; 8(1):55-58

A New and Efficient Enrichment Method for Metagenomic Sequencing of Monkeypox Virus

[Abstract] Background The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. Results A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. Conclusions Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.This work was supported by a grant from the SERGAS-Galician Healthcare Service (Program “Innova Saúde”) to GB, by CIBERINFEC and also by Instituto de Salud Carlos III (ISCIII) through the projects PI20/00413 to MP and PI21/00704 to G

Automation Proposal for the Intermediate Steps in the 16S FFPE Samples Analysis Pipeline

Cursos e Congresos, C-155[Abstract] In the day-to-day work of bioinformatics, the use of integrated software packages, which encompass a wide range of tools, enables the development of pipelines for omics data analysis. Within the various existing pipelines, we focus on the analysis of the 16S rRNA gene as it allows for the study of diversity and taxonomy of prokaryotic microorganisms such as Bacteria and Archaea. However, these pipelines often involve a sequence of multiple tools that require intermediate steps before further processing can proceed, as in the case between Cutadapt and DADA2. In fact, in a typical pipeline, the values for DADA2 input arguments ’trunc-len-f’ and ’trunc-len-r’ are extracted from the output of Cutadapt. The best approach for selecting optimal values (aka the trimming positions) is graphically visualizing Cutadapt output and manually selecting the most accurate trimming position length. Therefore, we propose the automation of this specific intermediate step between Cutadapt and DADA2 tools, by selecting values displayed in the graphs that meet the filtering criteria. This automation has been incorporated into a custom pipeline for the analysis of the microbiome in 16S paired-end samples from colorectal cancer patients, and could potentially serve as a standardization approach in these processesThe authors of this paper extend their sincere appreciation to the collaborative efforts and contributions of the meiGAbiome Group, aswell as the entire team of medical and anatomopathologists. Finally, we are deeply grateful to the patients whose selfless donations have made this and numerous other studies possibl

Quorum Sensing as a Target for Controlling Surface Associated Motility and Biofilm Formation in Acinetobacter Baumannii ATCC® 17978TM

[Abstract] The important nosocomial pathogen Acinetobacter baumannii presents a quorum sensing (QS) system (abaI/abaR) mediated by acyl-homoserine-lactones (AHLs) and several quorum quenching (QQ) enzymes. However, the roles of this complex network in the control of the expression of important virulence-related phenotypes such as surface-associated motility and biofilm formation is not clear. Therefore, the effect of the mutation of the AHL synthase AbaI, and the exogenous addition of the QQ enzyme Aii20J on surface-associated motility and biofilm formation by A. baumannii ATCC® 17978TM was studied in detail. The effect of the enzyme on biofilm formation by several multidrug-resistant A. baumannii clinical isolates differing in their motility pattern was also tested. We provide evidence that a functional QS system is required for surface-associated motility and robust biofilm formation in A. baumannii ATCC® 17978TM. Important differences were found with the well-studied strain A. nosocomialis M2 regarding the relevance of the QS system depending on environmental conditions The in vitro biofilm-formation capacity of A. baumannii clinical strains was highly variable and was not related to the antibiotic resistance or surface-associated motility profiles. A high variability was also found in the sensitivity of the clinical strains to the action of the QQ enzyme, revealing important differences in virulence regulation between A. baumannii isolates and confirming that studies restricted to a single strain are not representative for the development of novel antimicrobial strategies. Extracellular DNA emerges as a key component of the extracellular matrix in A. baumannii biofilms since the combined action of the QQ enzyme Aii20J and DNase reduced biofilm formation in all tested strains. Results demonstrate that QQ strategies in combination with other enzymatic treatments such as DNase could represent an alternative approach for the prevention of A. baumannii colonization and survival on surfaces and the prevention and treatment of infections caused by this pathogen.Xunta de Galicia; ED481A-2015/311Xunta de Galicia; IN606B-2019/010Biotechnology and Biological Sciences Research Council (United Kingdom); BB/R012415/1Xunta de Galicia; ED481A-2019/19

A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

<p>Abstract</p> <p>Background</p> <p><it>Acinetobacter baumannii </it>is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate <it>A. baumannii </it>genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of <it>A. baumannii</it>.</p> <p>Results</p> <p>We describe here a rapid and simple method of obtaining <it>A. baumannii </it>mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (<it>omp33</it>, <it>oxyR</it>, and <it>soxR</it>) by this method. In addition, we disrupted one of these genes (<it>omp33</it>) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining <it>A. baumannii </it>mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in <it>A. baumannii</it>.</p> <p>Conclusions</p> <p>This study provides a rapid and simple method of obtaining stable mutants of <it>A. baumannii </it>free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.</p

Characterization of a limestone in a batch fluidized bed reactor for sulfur retention under oxy-fuel operating conditions

12 FiguresCO2 and SO2 are some of the main polluting gases emitted into atmosphere in combustion processes using fossil fuel for energy production. The former is one of the major contributors to build-up the greenhouse effect implicated in global climate change and the latter produces acid rain. Oxy-fuel combustion is a technology, which consists in burning the fuel with a mix of pure O2 and recirculated CO2. With this technology the CO2 concentration in the flue gas may be enriched up to 95%, becoming possible an easy CO2 recovery. In addition, oxy-fuel combustion in fluidized beds allows in situ desulfurization of combustion gases by supplying calcium based sorbent.In this work, the effect of the principal operation variables affecting the sulfation reaction rate in fluidized bed reactors (temperature, CO2 partial pressure, SO2 concentration and particle size) under typical oxy-fuel combustion conditions have been analyzed in a batch fluidized bed reactor using a limestone as sorbent. It has been observed that sulfur retention can be carried out by direct sulfation of the CaCO3 or by sulfation of the CaO (indirect sulfation) formed by CaCO3 calcination. Direct sulfation and indirect sulfation operating conditions depended on the temperature and CO2 partial pressure. The rate of direct sulfation rose with temperature and the rate of indirect sulfation for long reaction times decreased with temperature. An increase in the CO2 partial pressure had a negative influence on the sulfation conversion reached by the limestone due to a higher temperature was needed to work in conditions of indirect sulfation. Thus, it is expected that the optimum temperature for sulfur retention in oxy-fuel combustion in fluidized bed reactors be about 925-950°C. Sulfation reaction rate rose with decreasing sorbent particle size and increasing SO2 concentration.This research has been supported by Spanish Ministry of Science and Innovation (MICINN, Project: CTQ2008-05399/PPQ) and by FEDER. M. de las Obras-Loscertales thanks MICINN for the F.P.I. fellowship.Peer Reviewe

Sulfur retention in an oxy-fuel bubbling fluidized bed combustor: Effect of coal rank, type of sorbent and O2 /CO2 ratio

In this work, SO2 retention via calcium-based sorbents added in a continuous bubbling fluidized bed combustor (~3 kWth) operating in oxy-fuel combustion mode is analyzed. Tests were performed at different operating temperatures with three sorbents, two limestones and one dolomite, and with three coals, ranging from lignite to anthracite, to analyze the influence of coal rank, type of sorbent, sorbent particle size, and O2/CO2 feeding ratio on the sulfation process It was found that the combustor temperature had a strong influence on the limestones sulfur retention with a maximum at 900-925 ºC. The behavior of the limestones was qualitatively similar with the three coals, attaining the highest sulfur retention values working with the lignite and the lowest working with the bituminous coal. On the contrary, with the dolomite the sulfur retention was hardly affected by the combustion temperature and the sulfur retentions attained were higher than with the limestones. The sulfur retention increased with diminishing the Ca-based sorbent particle size, and it was hardly affected by the O2/CO2 ratio fed into the combustor.This research has been supported by Spanish Ministry of Science and Innovation (MICINN, Project: CTQ2008-05399/PPQ) and by FEDER. M. de las Obras-Loscertales thanks MICINN for the F.P.I. fellowship and A. Rufas thanks CSIC for the JAE fellowship.Peer reviewe

Retención de SO2 durante la oxicombustión de carbón en lecho fluidizado y su influencia en otros gases contaminantes

La oxicombustión es una tecnología de captura de CO2 que consiste en quemar el combustible utilizando una mezcla de oxígeno puro y gas recirculado (principalmente compuesto por CO2 y vapor de H2O) proveniente de la salida del combustor. De esta manera, al no introducir aire al combustor se elimina la presencia de N2 en la corriente de salida, obteniendo finalmente una corriente altamente concentrada en CO2 y lista para su posterior transporte y confinamiento. Los primeros desarrollos de la tecnología de oxicombustión se han realizado en calderas de carbón pulverizado (CP). Sin embargo las calderas de lecho fluidizado, en especial las calderas de lecho fluidizado circulante (LFC), presentan ciertas ventajas como poder alimentar sorbentes cálcicos directamente al lecho para llevar a cabo in situ el proceso de retención del SO2 generado durante la combustión del carbón, siendo este tema el principal objeto de estudio en este trabajo. La reacción de sulfatación de los sorbentes cálcicos es una reacción sólido-gas entre el sorbente cálcico, CaCO3 o CaO (reactante sólido) y el SO2 y O2 (reactante gas) para formar un compuesto sólido y estable, CaSO4. Según las condiciones existentes en la caldera, en concreto la presión parcial de CO2 y la temperatura, el sorbente cálcico puede reaccionar con el SO2 y el O2 como CaO (en condiciones calcinantes) o como CaCO3 (en condiciones no calcinantes). En las calderas de combustión convencional con aire las condiciones de operación son siempre calcinantes, por lo que tiene lugar la sulfatación del sorbente calcinado. Sin embargo, en condiciones de oxicombustión, la presión parcial de CO2 en la caldera (60 -90 %CO2) es mayor que en la combustión con aire (15 %CO2), pudiendo existir condiciones calcinantes o no calcinantes. El objetivo principal de este trabajo es analizar el efecto de las principales variables de operación (relación O2/CO2 alimentada, tipo de carbón, tipo de sorbente y su tamaño de partícula, etc.) de los reactores de lecho fluidizado operando en condiciones de oxicombustión, para maximizar la retención del SO2 generado en la combustión de diferentes carbones mediante la adición de sorbentes cálcicos. Además, se ha analizado en detalle el efecto de la recirculación de los gases de combustión, típica de estos procesos, sobre las emisiones de otros gases contaminantes (SO2, NOx, Hg) a la salida del combustor. Para ello, se seleccionaron cuatro sorbentes cálcicos, tres calizas y una dolomía, a los que se les realizó una caracterización físico-química y estructural (porosimetría de Hg, SEM-EDX, fisisorción de N2, etc). Se observó que los sorbentes frescos, debido a su alto grado de cristalización, apenas tenían porosidad y que su superficie específica o área BET era despreciable. Sin embargo, cuando se encontraban calcinados, los cuatro sorbentes desarrollaban una estructura mesoporosa con una distribución de poros unimodal, aumentando notablemente su porosidad y superficie específica. Posteriormente, se analizó la reactividad y capacidad de sulfatación de los sorbentes cálcicos mediante análisis termogravimétrico (ATG) y en una instalación de lecho fluidizado (LF) discontinuo. En ATG se realizó un estudio de la etapa de calcinación mediante rampas de calentamiento a diferentes concentraciones de CO2. Estas pruebas experimentales permitieron conocer la existencia de unas condiciones de operación que aunque se correspondían con condiciones calcinantes, la velocidad de calcinación era tan lenta que se producía la sulfatación directa del CaCO3. Además, en LF discontinuo se estudió la influencia de las principales variables de operación, como la temperatura, la concentración de CO2, la concentración de SO2 y el tamaño de partícula, sobre la reacción de sulfatación de los sorbentes. Se observó que había un óptimo de temperatura con respecto a la retención de azufre en torno a los 900 ºC y que el efecto de la concentración de CO2 se podía considerar despreciable una vez definidas las condiciones de operación (calcinantes o no calcinantes). Asimismo, se observó que la conversión de sulfatación de los sorbentes aumentaba al disminuir su tamaño de partícula y al aumentar la concentración de SO2. En todos los casos se apreció como se alcanzaban mayores conversiones de sulfatación operando en condiciones calcinantes que en no calcinantes y que la velocidad de la reacción de sulfatación se llevaba a cabo en dos etapas con velocidades de reacción muy diferentes [1,2]. Una vez realizada la caracterización de los sorbentes cálcicos mediante ATG y lecho fluidizado discontinuo, se desarrolló un modelo cinético a nivel de partícula de la reacción de sulfatación para condiciones calcinantes [3]. El modelo de sulfatación desarrollado constaba de dos etapas de reacción. Una primera etapa donde la velocidad de reacción era rápida y estaba controlada por la difusión de SO2 y O2 a través del sistema poroso de la partícula hasta que se formaba una capa de producto, CaSO4, alrededor de la partícula debido al bloqueo de los poros más externos de la misma. A partir de ahí comenzaba la segunda etapa de reacción caracterizada por ser más lenta y por estar controlada por la difusión del gas reactante a través de la capa de producto, CaSO4, y que reaccionaba siguiendo el modelo de núcleo decreciente. Los parámetros cinéticos determinados a partir de este modelo predijeron adecuadamente las curvas de conversión de sulfatación ¿ tiempo obtenidas experimentalmente. A continuación se realizaron experimentos en una planta en continuo de lecho fluidizado burbujeante (LFB) de 3kWt operando en condiciones de oxicombustión. En total se llevaron a cabo más de 600 horas de operación. Se analizó el efecto de las principales variables de operación, como la concentración de oxígeno alimentado a la caldera, el tipo de carbón (antracita, lignito y carbón bituminoso), la reactividad y el tamaño de partícula del sorbente cálcico y el efecto de la recirculación de los gases de combustión (SO2, NO y vapor de H2O), sobre el proceso de retención de SO2, así como también su influencia sobre otros gases contaminantes como NOx y Hg. Se observó que las variables que más influían en el proceso de retención de SO2 eran la temperatura (condiciones calcinantes y no calcinantes), el tipo y tamaño de partícula de sorbente y la recirculación de SO2 [4-6]. Asimismo, también se encontró que la recirculación de vapor de agua tenía una gran influencia sobre la reducción de la concentración de NO a la salida de la caldera. Finalmente, se realizó el modelado del proceso de retención de SO2 en un LFC a escala semi-industrial incluyendo el modelo de sulfatación a nivel de partícula y los parámetros cinéticos determinados anteriormente. Se simuló, sobre el proceso de retención de SO2, el efecto de las principales variables de operación de este tipo de combustores, tales como la temperatura, la reactividad y tamaño de partícula del sorbente cálcico, tipo de carbón, la relación molar Ca/S, la concentración de oxígeno alimentada al combustor, el tiempo de residencia de los sólidos y limpieza de gases de recirculación. El modelo desarrollado permitió optimizar el proceso de retención de SO2 en LFC operando en condiciones de oxicombustión. Bibliografía. [1] Calcium-based sorbents behaviour during sulphation at oxy-fuel fluidised bed combustion conditions. F. García-Labiano, A. Rufas, L.F. de Diego, M. de las Obras-Loscertales, P. Gayán, A. Abad, J. Adánez. Fuel 90 (2011) 3100-3108. [2] Characterization of a limestone in a batch fluidized bed reactor for sulfur retention under oxy-fuel operating conditions. L.F. de Diego, M. de las Obras-Loscertales, F. García-Labiano, A. Rufas, A. Abad, P. Gayán, J. Adánez. International Journal of Greenhouse Gas Control 5 (2011) 1190-1198. [3] Modelling of limestone sulfation for oxy-fuel fluidized bed combustion conditions. M. de las Obras-Loscertales, L.F. de Diego, F. García-Labiano, A. Rufas, A. Abad, P. Gayán, J. Adánez. Energy & fuels 27 (4) (2013) 2266-2274. [4] Optimum temperature for sulphur retention in fluidised beds working under oxy-fuel combustion conditions. L.F. de Diego, A. Rufas, F. García-Labiano, M. de las Obras-Loscertales, A. Abad, P. Gayán, J. Adánez. Fuel 114 (2013) 106-113. [5] Pollutant emissions in a bubbling fluidized bed combustor working in oxy-fuel operating conditions. Effect of flue gas recirculation. L.F. de Diego, M. de las Obras-Loscertales, A. Rufas, F. García-Labiano, P. Gayán, A. Abad, J. Adánez. Applied Energy 102 (2013) 860-867. [6] Sulfur retention in an oxy-fuel bubbling fluidized bed combustor: Effect of coal rank, type of sorbent and O2/CO2 ratio. M. de las Obras-Loscertales, L.F. de Diego, F. García-Labiano, A. Rufas, A. Abad, P. Gayán, J. Adánez. Fuel 137 (2014) 384-392