Diseño y evaluación de un procedimiento on-line para la biorremediación de aguas radiactivas en centrales nucleares

Recientemente se ha demostrado la existencia de microorganismos en las piscinas de almacenamiento de combustible nuclear gastado en las centrales nucleares utilizando técnicas convencionales de cultivo en el laboratorio. Estudios posteriores han puesto de manifiesto que los microorganismos presentes eran capaces de colonizar las paredes de acero inoxidable de las piscinas formando biopelículas. Adicionalmente se ha observado la capacidad de estas biopelículas de retener radionúclidos, lo que hace pensar en la posibilidad de utilizarlas en la descontaminación de las aguas radiactivas de las piscinas. En la presente tesis se plantea conocer más profundamente la biodiversidad microbiana de las biopelículas utilizando técnicas de biología molecular como la clonación, además de desarrollar un sistema de descontaminación a escala piloto con el objetivo de valorar si el proceso podría resultar escalable a nivel industrial. Para ello se diseñaron y fabricaron dos biorreactores en acero inoxidable compatibles con las condiciones específicas de seguridad sísmica y protección frente a la radiación en la zona controlada de una central nuclear. Los biorreactores se instalaron en la Central Nuclear de Cofrentes (Valencia) en las proximidades de las piscinas de almacenamiento de combustible nuclear gastado y precediendo a las resinas de intercambio iónico, de forma que reciben el agua de las piscinas permitiendo el análisis in situ de la radiación eliminada del agua de las mismas. Se conectó una lámpara de luz ultravioleta a uno de los biorreactores para poder comparar el desarrollo de bipelículas y la retención de radiactividad en ambas condiciones. En estos biorreactores se introdujeron ovillos de acero inoxidable y de titanio que se extrajeron a diversos tiempos, hasta 635 días para los ovillos de acero inoxidable y hasta 309 días para los ovillos de titanio. Se analizaron las biopelículas desarrolladas sobre los ovillos por microscopía electrónica de barrido y por microscopía de epifluorescencia. Se extrajo el ADN de las biopelículas y, tras su clonación, se identificaron los microorganismos por técnicas independientes de cultivo. Asimismo se determinó por espectrometría gamma la capacidad de las biopelículas para retener radionúclidos. Los microorganismos radiorresistentes identificados pertenecen a los grupos filogenéticos Alpha-proteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus y Bacteroidetes. Las secuencias de estos microorganismos se han depositado en el GenBank con los números de acceso KR817260-KR817405. Se ha observado una distribución porcentual ligeramente diferente en relación con el tipo de biorreactor. Las biopelículas han retenido fundamentalmente radionúclidos de activación. La suma de Co-60 y Mn-54 ha llegado en ocasiones al 97%. Otros radionúclidos retenidos han sido Cr-51, Co-58, Fe-59, Zn-65 y Zr-95. Se sugiere un mecanismo del proceso de retención de radionúclidos relacionado con el tiempo de formación y desaparición de las biopelículas. Se ha valorado que el proceso escalable puede ser económicamente rentable. ABSTRACT The existence of microorganisms in spent nuclear fuel pools has been demonstrated recently in nuclear power plants by using conventional microbial techniques. Subsequent studies have revealed that those microorganisms were able to colonize the stainless steel pool walls forming biofilms. Additionally, it has been observed the ability of these biofilms to retain radionuclides, which suggests the possibility of using them for radioactive water decontamination purposes. This thesis presents deeper knowledge of microbial biofilms biodiversity by using molecular biology techniques such as cloning, and develops a decontamination system on a pilot scale, in order to assess whether the process could be scalable to an industrial level. Aiming to demonstrate this was feasible, two stainless steel bioreactors were designed and manufactured, both were compatible with seismic and radiation protection standards in the controlled zone of a nuclear plant. These bioreactors were installed in the Cofrentes Nuclear Power Plant (Valencia) next to the spent nuclear fuel pools and preceding (upstream) ion exchange resins. This configuration allowed the bioreactors to receive water directly from the pools allowing in situ analysis of radiation removal. One ultraviolet lamp was connected to one of the bioreactors to compare biofilms development and radioactivity retention in both conditions. Stainless steel and titanium balls were introduced into these bioreactors and were removed after different time periods, up to 635 days for stainless steel balls and up to 309 days for titanium. Biofilms developed on the balls were analyzed by scanning electron microscopy and epifluorescence microscopy. DNA was extracted from the biofilms, was cloned and then the microorganisms were identified by independent culture techniques. Biofilms ability to retain radionuclides was also determined by gamma spectrometry. The identified radioresistant organisms belong to the phylogenetic groups Alphaproteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus and Bacteroidetes. The sequences of these microorganisms have been deposited in GenBank (access numbers KR817260-KR817405). A different distribution of microorganisms was observed in relation to the type of bioreactor. Biofilms have essentially retained activation radionuclides. Sometimes the sum of Co-60 and Mn-54 reached 97%. Cr-51, Co-58, Fe-59, Zn-65 and Zr-95 have also been retained. A radionuclide retention process mechanism related to biofilms formation and disappearance time is suggested. It has been assessed that the scalable process can be economically profitable.

Compatibilidad de Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) y Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), importantes enemigos naturales de la mosca blanca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) en cultivos hortícolas, con nuevas barreras físicas selectivas y modernos plaguicidas

Los programas de Gestión Integrada de Plagas (GIP) promueven el uso de estrategias de control que sean respetuosas con el medio ambiente, sin embargo el uso de insecticidas en los cultivos hortícolas sigue siendo necesario para el control de determinadas plagas, como es el caso de la mosca blanca Bemisia tabaci (Gennadius). Por ello, el objetivo de esta tesis es el estudio de la integración de las tres estrategias de control más empleadas hoy en día para el control de plagas: el control biológico, el físico y el químico. Una primera parte de este trabajo ha consistido en el estudio de los efectos letales y subletales de once insecticidas, aplicados a la dosis máxima de campo, sobre los enemigos naturales Eretmocerus mundus Mercet y Amblyseius swirskii Athias-Henriot, mediante ensayos de laboratorio y persistencia (laboratorio extendido). Para la evaluación de la toxicidad de los insecticidas sobre los estados de vida más protegidos de estos enemigos naturales, se trataron bajo la Torre de Potter las pupas de E. mundus y los huevos de A. swirskii. Además, se llevaron a cabo ensayos de contacto residual para determinar los efectos letales y subletales de estos insecticidas sobre el estado adulto de ambas especies de enemigos naturales. Para ello, los pesticidas se aplicaron sobre placas de cristal (laboratorio) o sobre plantas (laboratorio extendido: persistencia). Los resultados mostraron que los insecticidas flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen y spirotetramat eran compatibles con el estado de pupa de E. mundus (OILB 1: Inocuos). Sin embargo, abamectina, deltametrina y emamectina fueron categorizadas como ligeramente tóxicas (OILB 2) al causar efectos deletéreos. Los dos pesticidas más tóxicos fueron spinosad y sulfoxaflor, los cuales redujeron significativamente la emergencia de las pupas tratadas (OILB 4: Tóxicos). Flonicamida, flubendiamida, metoxifenocida y spiromesifen fueron compatibles con el estado adulto de E. mundus (OILB 1: Inocuos). Abamectina, deltametrina, emamectina, metaflumizona y spiromesifen pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Al contrario, spinosad y sulfoxaflor no resultaron ser compatibles (OILB D: Persistentes), aunque la realización de ensayos adicionales es necesaria para ver los efectos de los mismos en campo. Todos los insecticidas estudiados, excepto el spirotetramat (OILB 2: Ligeramente tóxico), fueron selectivos para el estado de huevo de A. swirskii (OILB 1: Inocuos). Flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen, spirotetramat y sulfoxaflor, fueron compatibles con el estado adulto de A. swirskii (OILB 1: Inocuos). Abamectina, deltametrina, emamectina y spinosad pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Entre las nuevas estrategias de la GIP, los plásticos y mallas fotoselectivas han demostrado ser una herramienta importante para el control de plagas y enfermedades en cultivos hortícolas protegidos. Por ello, en una segunda parte de este trabajo, se estudiaron tanto los efectos directos, como la combinación de efectos directos y mediados por planta y plaga de ambientes pobres en luz UV, en presencia o ausencia del Virus del rizado amarillo del tomate (TYLCV), sobre E. mundus. En primer lugar, se realizó un ensayo al aire libre para la evaluación de la capacidad de vuelo de E. mundus en cajas tipo túnel (1 x 0,6 x 0,6 m) cubiertas con distintas barreras absorbentes de luz UV. Se detectó un efecto directo en la capacidad de orientación de E. mundus, debido a que este parasitoide utiliza estímulos visuales para localizar a sus huéspedes, únicamente en las barreras que bloqueaban más del 65% de la luz UV (malla G). En segundo lugar, bajo condiciones de invernadero, se evaluó la combinación de efectos directos y mediados por planta y plaga sobre E. mundus, usando plantas de tomate sanas o infectadas con el TYLCV y cajas (30 x 30 x 60 cm) cubiertas con los distintos plásticos fotoselectivos. En este caso, no se observó ningún efecto en la capacidad benéfica del parasitoide cuando este estaba en contacto con plantas de tomate infestadas con ninfas de B. tabaci, lo que demuestra que este insecto usa estímulos táctiles para encontrar a sus huéspedes a cortas distancias. Además, las diferentes condiciones de radiación UV estudiadas tuvieron cierto impacto en la morfología, fisiología y bioquímica de las plantas de tomate, infestadas o no con el virus de la cuchara, detectándose pequeñas alteraciones en alguno de los parámetros estudiados, como el peso fresco y seco, el contenido en H y el espesor de las cutículas y de las paredes celulares de la epidermis foliar. Por último, no se observaron efectos de la radiación UV mediados por planta, ni en B. tabaci ni en su parasitoide, E. mundus. En una tercera parte, se evaluaron los efectos de una malla tratada con bifentrin sobre ambos enemigos naturales, en ensayos de laboratorio, semicampo y campo. Las mallas tratadas fueron diseñadas originariamente para el control de mosquitos vectores de la malaria, y actualmente se está trabajando para su uso en agricultura, como una nueva estrategia de control de plagas. En ensayos de laboratorio, cuando adultos de E. mundus y A. swirskii se expusieron por contacto durante 72 horas con la malla tratada (cajas de 6 cm diámetro), se registró una alta mortalidad. Sin embargo, en el ensayo de preferencia, estos enemigos naturales no fueron capaces de detectar la presencia de bifentrin y, en aquellos individuos forzados a atravesar la malla tratada, no se observó mortalidad a corto plazo (72 horas). En estudios de semicampo, llevados a cabo bajo condiciones de invernadero en cajas de 25 x 25 x 60 cm de altura, la capacidad benéfica de E. mundus no se vio afectada. Finalmente, en ensayos de campo llevados a cabo en invernaderos comerciales (4000m2) en Almería, A. swirskii no se vio afectado por la presencia en el cultivo de la malla tratada con bifentrin y los niveles de infestación de B. tabaci y F. occidentalis detectados bajo dicha malla, fueron inferiores a los del control. Por último, se ha evaluado la composición de la microflora bacteriana de tres especies de parasitoides, E. mundus, Eretmocerus eremicus Rose & Zolnerowich y Encarsia formosa Gahan, y la influencia de la misma en su susceptibilidad a insecticidas. Se llevó a cabo una extracción total de ADN de los insectos y la región variable V4 del ARNr se amplificó usando cebadores universales bacterianos. Para identificar las secuencias de los géneros bacterianos presentes en los parasitoides, se realizó una Next Generation sequencing (Illumina sequencing). Una vez identificados los géneros bacterianos, el gen ADNr 16S de las Actinobacterias se amplificó del ADN extraído de los insectos, usando cebadores universales bacterianos y específicos de Actinobacterias, y los productos de la Nested PCR fueron clonados para identificar todas las especies del género Arthrobacter. Tres bacterias (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi y A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), próximas a las especies de Arthrobacter presentes en los parasitoides, se obtuvieron de la colección bacteriana del BCCMTM/LMG y se midió su actividad esterasa. Finalmente, se realizaron ensayos con antibióticos (tetraciclina) y de contacto residual con insecticidas (abamectina) para determinar la influencia de las especies de Arthrobacter en la susceptibilidad de E. mundus a insecticidas. Los resultados muestran que este género bacteriano puede afectar a la toxicidad de E. mundus a abamectina, mostrando la importancia de la comunidad microbiana en enemigos naturales, factor que debe ser considerado en los estudios de evaluación de los riesgos de los insecticidas. ABSTRACT Integrated Pest Management (IPM) programs promote the use of control strategies more respectful with the environment; however the use of insecticides in vegetable crops is still needed to control certain pests, such as the whitefly Bemisia tabaci (Gennadius). Therefore, the objective of this work is to study the integration of the three most commonly used pest control strategies nowadays: biological, physical and chemical control. Firstly, the lethal and sublethal effects of eleven insecticides, applied at their maximum field recommended concentration, on the parasitic wasp Eretmocerus mundus Mercet and the predator Amblyseius swirskii Athias-Henriot has been assessed in the laboratory and in persistence tests (extended laboratory). To test the effects of pesticides on the most protected life stage of these natural enemies, E. mundus pupae and A. swirskii eggs were sprayed under a Potter precision spray tower. Laboratory contact tests were therefore conducted to determine the lethal and sublethal effects of these pesticides on the adult stage of these natural enemies. In the residual contact tests the pesticides were applied on glass plates (laboratory) or plants (extended laboratory: persistence). The study showed that the insecticides flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen and spirotetramat were selective for E. mundus pupae (IOBC 1: Harmless). Nevertheless, abamectin, deltamethrin and emamectin were categorized as slightly harmful (IOBC 2) due to the deleterious effects caused. The two most harmful pesticides were spinosad and sulfoxaflor, which significantly reduced the adult emergence from treated pupae (IOBC 4: Harmful). Flonicamid, flubendiamide, methoxyfenozide and spiromesifen were compatible with E. mundus adults (IOBC 1: Harmless). Base on the duration of the harmful activity, abamectin, deltamethrin, emamectin, metaflumizone and spirotetramat could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. On the contrary, spinosad and sulfoxaflor were not compatible (IOBC D: persistent), although additional studies are required to determine their effects under field conditions. All the pesticides tested, except spirotetramat (IOBC 2: Slightly harmful), were selective for A. swirskii eggs (IOBC 1: Harmless). Flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen, spirotetramat and sulfoxaflor were compatible with A. swirskii adults (IOBC 1: Harmless). However, abamectin, deltamethrin, emamectin and spinosad could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. Among new IPM strategies, UV-absorbing photoselective plastic films and nets have been shown to be an important tool for the control of pests and diseases in horticultural protected crops. Because of that, we secondly studied the plant and pest insect-mediated and/or the direct effects on E. mundus under different UV radiation conditions, in presence or absence of the Tomato Yellow Leaf Curl Virus (TYLCV). In the first experiment, performed outdoors, the flight activity of E. mundus was studied in one-chamber tunnels (1 x 0.6 x 0.6 m) covered with different photoselective barriers. Because E. mundus uses visual cues for host location at a long distance, a direct effect on its host location ability was detected, but only in the UV-absorbing barriers blocking more than 65% of the UV light (G net). In a second experiment, the direct and plant and pest insect-mediated effects of different UV radiation conditions on E. mundus were studied, inside cages (30 x 30 x 60 cm) covered with the different UVplastic films and under greenhouse conditions, using healthy or TYLCV-virus infected tomato plants. In this case, not any effect on the beneficial capacity of this parasitoid was detected, proving that he uses tactile cues at a short distance of the host. Moreover, the different UV radiation conditions studied had a certain direct impact in the morphology, physiology and biochemistry of tomato plants infested or not with the TYLCV, and small alterations in some parameters such as fresh and dry weight, H percentage and cuticle and cell wall thickness of epidermal cells of the leaves, were detected. Finally, none plant-mediated UV effects neither in the whitefly B. tabaci nor in their parasitic wasp were found. Thirdly, the effects of a bifenthrin treated net were evaluated in different laboratory, semi-field and field experiments on the natural enemies studied. Treated nets were developed long time ago aiming at the control of the mosquitoes vectors of malaria, and nowadays, there is a great interest on assessing the possibility of their use in agriculture. In laboratory assays, a high mortality was recorded when E. mundus and A. swirskii adults were exposed by contact to the bifenthrin treated net for 72 hours in small cages (12 cm diameter). However, these natural enemies were not able to detect the presence of bifenthrin in a dual-choice test and no short-term mortality (72 hours) was recorded in those individuals that went through the treated net. In semi-field assays, performed under greenhouse conditions with cages of 25 x 25 x 60 cm high, the beneficial capacity of E. mundus was not affected. Finally, in field assays carried out in commercial multispan greenhouses (4000 m2) in Almería, A. swirskii was not affected by the presence of the bifenthrin treated net in the crop and the B. tabaci and F. occidentalis infestation levels were significantly lower than in the control. Finally, the composition of the microflora present in three species of parasitoids, E. mundus, Eretmocerus eremicus Rose & Zolnerowich and Encarsia formosa Gahan, and its influence in their susceptibility to insecticides, have been assessed. A total DNA extraction was performed on insects and universal bacterial primers were used to amplify the variable V4 region of the rRNA. A Next Generation sequencing (Illumina sequencing) was performed to identify the sequences of the bacterial genera present in the parasitic wasps. Once, the bacterial genera were identified, 16S rDNA gene of Actinobacteria were amplified from insects DNA extracts using the universal bacterial and actinobacterial primers, and the nested PCR products, were cloned to identify the Arthrobacter species. Three bacteria (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi and A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), having the closest match with the Arthrobacter species present in the parasitic wasps, were obtained from the BCCMTM/LMG bacteria collection and its esterase activity was measured. Finally, antibiotic and residual contact tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides (abamectin). The results suggest that this bacterial genus can affect the toxicity of E. mundus to abamectin, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides.

Culturable aerobic and facultative bacteria from the gut of the polyphagic dung beetle Thorectes lusitanicus Jeckel

Unlike other dung beetles, the Iberian geotrupid Thorectes lusitanicus exhibits polyphagous behavior; for example, it is able to eat acorns, fungi, fruits, and carrion in addition to the dung of different mammals. This adaptation to digest a wider diet has physiological and developmental advantages and requires key changes in the composition and diversity of the beetle's gut microbiota. In this study, we isolated aerobic, facultative anaerobic, and aerotolerant microbiota amenable to grow in culture from the gut contents of T. lusitanicus and resolved isolate identity to the species level by sequencing 16S rRNA gene fragments. Using BLAST similarity searches and maximum likelihood phylogenetic analyses, we were able to reveal that the analyzed fraction (culturable, aerobic, facultative anaerobic, and aerotolerant) of beetle gut microbiota is dominated by the phyla Proteobacteria, Firmicutes and Actinobacteria. Among Proteobacteria, members of the order Enterobacteriales (Gammaproteobacteria) were the most abundant. The main functions associated with the bacteria found in the gut of T. lusitanicus would likely include nitrogen fixation, denitrification, detoxification, and diverse defensive roles against pathogens.

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.

Geochemical and Microbiological Studies of Nitrous Oxide Variations within the New NEEM Greenland Ice Core During the Last Glacial Period

Deep polar ice cores provide atmospheric records of nitrous oxide (N₂O) and other trace gases reflecting climate history along with a parallel archive of microbial cells transported with mineral dust, marine and volcanic aerosols from around the globe. Our interdisciplinary study of 32 samples from different depths of the recently drilled NEEM Greenland ice core addressed the question whether the identified microorganisms were capable of post-depositional biological production of N₂O in situ. We used high-resolution geochemical and microbiological approaches to examine the N₂O concentrations, the quantitative distributions of dust, Ca⁺², NH₄⁺ and NO₃⁻ ¡ons related to N cycle pathways, the microbial abundance and diversity at specific NEEM core depths from 1758 m to 1867.8 m. Results showed varying concentrations of N₂O (220 –271.5 ppb). Microbial abundance fluctuated between 3.3 x 10⁴ and 3.3 x 10⁶ cells mL⁻¹ in direct correlation with dust and Ca²⁺ concentrations with higher cell numbers deposited during colder periods. The average values of NH₄⁺ and NO₃⁻ indicated that substrates were available for the microorganisms capable of utilizing them. PCR amplification of selected functional genes involved in bacterial and archaeal nitrification and denitrification was not successful. Sanger and Illumina MiSeq sequence analyses of SSU rRNA genes showed variable representation of Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, chloroplasts and fungi. The metabolic potential of the dominant genera of Proteobacteria and Firmicutes as possible N₂O producers suggested that denitrification activity may have led to in-situ production and accumulation of N₂O.

Table 2: Bacterial diversity in Ny-Ålesund, Spitzbergen

We describe the antibiotic resistance profiling of bacterial isolates collected from Ny-Alesund, Arctic, as part of the Indian Arctic Summer Expedition 2009. It was interesting to note that the bacterial isolates collected from the Arctic showed multidrug resistance. 32% of the isolates were found to be multi- drug resistant with several combinations of antibiotics. The 16S rRNA sequencing results shows a diverse group of bacteria belonging to Phyla Proteobacteria, Actinobacteria and Bacteriodetes and their relatedness was studied by phylogenetic analysis. While analysing the plasmid profiling, the most resistant two strains of Pseudomonas migulae showed multiple plasmids of varying sizes ~5.2-5.3 kb and ~9.5 kb. The extent and frequency of multidrug resistance in the polar bacteria deserves close monitoring and efforts to understand the various molecular mechanisms of drug resistance and control the spread of antibiotic resistance in polar environment is called for.

Culturable bacterial symbionts isolated from two distinct sponge species (Pseudoceratina clavata and Rhabdastrella globostellata) from the Great Barrier Reef display similar phylogenetic diversity

The diversity of the culturable microbial communities was examined in two sponge species-Pseudoceratina clavata and Rhabdastrella globostellata. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis. The bacterial community structures represented in both sponges were found to be similar at the phylum level by the same four phyla in this study and also at a finer scale at the species level in both Firmicutes and Alphaproteobacteria. The majority of the Alphaproteobacteria isolates were most closely related to isolates from other sponge species including alpha proteobacterium NW001 sp. and alpha proteobacterium MBIC3368. Members of the low %G + C gram-positive (phylum Firmicutes), high %G + C gram-positive (phylum Actinobacteria), and Cytophaga-Flavobacterium-Bacteroides (phylum Bacteroidetes) phyla of domain Bacteria were also represented in both sponges. In terms of culturable organisms, taxonomic diversity of the microbial community in the two sponge species displays similar structure at phylum level. Within phyla, isolates often belonged to the same genus-level monophyletic group. Community structure and taxonomic composition in the two sponge species P. clavata and Rha. globostellata share significant features with those of other sponge species including those from widely separated geographical and climatic regions of the sea.

Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches

Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata. Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of culturable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metagenomes derived from other sponge species from other geographical regions.

Isolates of 'Candidatus Nostocoida limicola' Blackall et al. 2000 should be described as three novel species of the genus Tetrasphaera, as Tetrasphaera jenkinsii sp nov., Tetrasphaera vanveenii sp nov and Tetrasphaera veronensis sp nov.

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (> 94 %) between strains of the filamentous bacterium 'Candidatus Nostocoida limicola' and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S-23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria. This suggestion receives additional support from DNA-DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that 'Candidatus N. limicola' strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74(T) = DSM 17519(T) = NCIMB 14128(T)), 'Candidatus N. limicola' strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70(T) = DSM 17518(T) = NCIMB 14127(T)) and 'Candidatus N. limicola' strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1(T) = DSM 17520(T) = NCIMB 14129(T)).

Changes in Community Structure of Sediment Bacteria Along the Florida Coastal Everglades Marsh–Mangrove–Seagrass Salinity Gradient

Community structure of sediment bacteria in the Everglades freshwater marsh, fringing mangrove forest, and Florida Bay seagrass meadows were described based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) patterns of 16S rRNA gene fragments and by sequencing analysis of DGGE bands. The DGGE patterns were correlated with the environmental variables by means of canonical correspondence analysis. There was no significant trend in the Shannon–Weiner index among the sediment samples along the salinity gradient. However, cluster analysis based on DGGE patterns revealed that the bacterial community structure differed according to sites. Not only were these salinity/vegetation regions distinct but the sediment bacteria communities were consistently different along the gradient from freshwater marsh, mangrove forest, eastern-central Florida Bay, and western Florida Bay. Actinobacteria- and Bacteroidetes/Chlorobi-like DNA sequences were amplified throughout all sampling sites. More Chloroflexi and members of candidate division WS3 were found in freshwater marsh and mangrove forest sites than in seagrass sites. The appearance of candidate division OP8-like DNA sequences in mangrove sites distinguished these communities from those of freshwater marsh. The seagrass sites were characterized by reduced presence of bands belonging to Chloroflexi with increased presence of those bands related to Cyanobacteria, γ-Proteobacteria, Spirochetes, and Planctomycetes. This included the sulfate-reducing bacteria, which are prevalent in marine environments. Clearly, bacterial communities in the sediment were different along the gradient, which can be explained mainly by the differences in salinity and total phosphorus.

Metagenoma de comunidades microbianas expostas à radiação natural e a metais

Many species have specialized to live in the most varied existing environments showing the remarkable adaptability of the microbial world the most diverse physicochemical conditions. Environments exposed to natural radiation and metals are scarce around the world, presenting a microbiota still unknown. With a total number estimated between 4 and 6 x 1030 microrganisms on earth, they constitute an enormous biological and genetic pool to be explored. Metagenomic approach independent of cultivation, provides a new form to access to the potential genomic environmental samples becoming a powerful tool for the elucidation of ecological functions, metabolic profiles, as well as to identify new biomolecules. In this context, the genetic material of environmental soil and water samples from Açude Boqueirao Parelhas-RN, under the influence of natural radiation and the presence of metals, was extracted, pirosequencing and the generated sequences were analyzed by bioinformatics programs (MG-RAST and STAMP). Taxonomic comparative profiles of both samples showed high abundance of Domain Bacteria, followed by a small portion attributable to Eucaryota Domains, Archaea and Viruses. Proteobacteria, Actinobacteria and Bacterioidetes phyla showed the greater dominance in both samples. Important genera and species associated with resistance to various stressors found in region were observed. Sequences related to oxidative and heat stress, DNA replication and repair, and resistance to toxic compounds were observed, suggesting a significant relationship between the microbiota and their metabolic profile, influenced by regional environmental variables. The results of this study add valuable and unpublished data on the composition of microbial communities in these regions

Selected soil properties and bacterial abundance in the glacier forefields 'Black Valley Transect' and 'Glacier Transect' on Larsemann Hills, East Antarctica

Antarctic glacier forefields are extreme environments and pioneer sites for ecological succession. Increasing temperatures due to global warming lead to enhanced deglaciation processes in cold-affected habitats, and new terrain is becoming exposed to soil formation and microbial colonization. However, only little is known about the impact of environmental changes on microbial communities and how they develop in connection to shifting habitat characteristics. In this study, using a combination of molecular and geochemical analysis, we determine the structure and development of bacterial communities depending on soil parameters in two different glacier forefields on Larsemann Hills, East Antarctica. Our results demonstrate that deglaciation-dependent habitat formation, resulting in a gradient in soil moisture, pH and conductivity, leads to an orderly bacterial succession for some groups, for example Cyanobacteria, Bacteroidetes and Deltaproteobacteria in a transect representing 'classical' glacier forefields. A variable bacterial distribution and different composed communities were revealed according to soil heterogeneity in a slightly 'matured' glacier forefield transect, where Gemmatimonadetes, Flavobacteria, Gamma- and Deltaproteobacteria occur depending on water availability and soil depth. Actinobacteria are dominant in both sites with dominance connected to certain trace elements in the glacier forefields.

Microbial community composition and bacterioplankton at time series station Helgoland Roads, North Sea

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. This study explores such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. The surface water samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' (54° 11.3' N, 7° 54.0' E) between the main island and the minor island, Düne (German for 'dune') using small research vessels (http://www.awi.de/en/expedition/ships/more-ships.html). Water depths at this site fluctuate from 6 to 10 m over the tidal cycle. Samples were processed as described previously (Teeling et al., 2012; doi:10.7554/eLife.11888.001) in the laboratory of the Biological Station Helgoland within less than two hours after sampling. Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously (Thiele et al., 2011; doi:10.1016/B978-0-444-53199-5.00056-7). To obtain total cell numbers, DNA of formaldehyde fixed cells filtered on 0.2 mm pore sized filters was stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescently labeled cells were subsequently counted on filter sections using an epifluores-cence microscope. Likewise, bacterioplankton community composition was assessed by catalyzedreporter deposition fluorescence in situ hybridization (CARD-FISH) of formaldehyde fixed cells on 0.2 mm pore sized filters.