Evaluación in vitro de la capacidad biorremediadora de hongos filamentosos sobre petróleo crudo / Bioremediative Capacity Evaluation in vitro of the filamentous fungi on crude oil

Resumen Objetivo. Evaluar la capacidad biorremediadora de algunas especies de hongos filamentosos en suelos contaminados con petróleo crudo. Métodos. Se aislaron 138 cepas a partir de 90 cultivos primarios en muestras de suelos obtenidas en los municipios de Yondó (Antioquia), Acacías (Meta) y Tumaco (Nariño), se identificaron las especies por medio de claves taxonómicas y la amplificación por PCR de la región ITS ubicándose en siete géneros de hongos filamentosos. Del total de las especies se escogieron tres para el experimento que fueron: Neosartorya sp. Cepa A/N -1, Aspergillus sp. Cepa Y/As-3 y Rhizomucor sp. Cepa 1A/R-1; se realizó la micorremediación con la técnica de Landfarming modificado al diseñarse un microcosmos con 50g de suelo contaminado con petróleo crudo °API de 21.6 a concentraciones de 20.000 ppm y 30.000ppm para cada una de las especies en un inóculo de 300 conidios/ml en agua destilada, ajustado en cámara de Neubauer y las tres especies en consorcio en suelo contaminado con petróleo crudo °API de 21.6 a concentraciones de 40.000ppm, 60.000ppm, 80.000ppm y 100.000ppm con un inóculo de 300 conidios/ml. Se valoró el proceso por espectofotometría. Además se estableció las enzimas empleadas en la micorremediación y la citotoxicidad de las cepas empleadas. Resultado. La micorremediación realizada por las especies individuales fue en promedio entre 2 y 6 días y cuando se aplicaron las tres especies en consorcio se realizó en un promedio entre 6 y 10 días, la enzima empleada es peroxidasa y la citotoxicidad es negativa.

Abstract Objective. Evaluate the bioremediation capacity of some species of filamentous fungi in soils contaminated with crude oil. Methods. 138 strains were isolated from 90 primary culture in soil samples obtained in the municipalities of Yondó (Antioquia), Acacías (Meta) and Tumaco (Nariño), species were identified by means of taxonomic keys and PCR amplification of the ITS region located in seven genera of filamentous fungi. Of the total of the species, three were chosen for the experiment, which were: Neosartorya sp. Cepa A/N- 1, Aspergillus sp. Cepa Y/As -3 and Rhizomucor sp. Cepa 1A/R-1; Micoremediation was performed with the modified Landfarming technique when designing a microcosm with 50g of soil contaminated with crude oil API 21.6 at concentrations of 20,000 ppm and 30,000ppm for each of the species in an inoculum of 300 conidia / ml in water distilled, adjusted in Neubauer chamber and the three species in consortium in soil contaminated with crude oil API 21.6 at concentrations of 40,000ppm, 60,000ppm, 80,000ppm and 100,000ppm with an inoculum of 300 conidia / ml. The process was evaluated by spectrophotometry. In addition, the enzymes used in the micoremediation and cytotoxicity of the strains used were established. Result. The micoremediation performed by the individual species was on average between 2 and 6 days and when the three species were applied in a consortium it was carried out on average between 6 and 10 days, the enzyme used is peroxidase and the cytotoxicity is negative.

Characterization of Rhodococcus sp. A5wh isolated from a high altitude Andean lake to unravel the survival strategy under lithium stress / Caracterización de las estrategias seguidas por Rhodococcus sp. A5wh proveniente de laguna de altura en su adaptación a la presencia de litio

Lithium (Li) is widely distributed in nature and has several industrial applications. The largest reserves of Li (over 85%) are in the so-called "triangle of lithium" that includes the Salar de Atacama in Chile, Salar de Uyuni in Bolivia and Salar del Hombre Muerto in Argentina. Recently, the use of microorganisms in metal recovery such as copper has increased; however, there is little information about the recovery of lithium. The strain Rhodococcus sp. A5wh used in this work was previously isolated from Laguna Azul. The assays revealed that this strain was able to accumulate Li (39.52% of Li/g microbial cells in 180 min) and that it was able to grow in its presence up to 1 M. In order to understand the mechanisms implicated in Li tolerance, a proteomic approach was conducted. Comparative proteomic analyses of strain A5wh exposed and unexposed to Li reveal that 17 spots were differentially expressed. The identification of proteins was performed by MALDI-TOF/MS, and the obtained results showed that proteins involved in stress response, transcription, translations, and metabolism were expressed under Li stress. This knowledge constitutes the first proteomic approach to elucidate the strategy followed by Rhodococcus to adapt to Li.

El litio (Li) es un elemento químico con múltiples aplicaciones industriales. Es considerado uno de los minerales más ampliamente distribuidos en la naturaleza. Sus mayores reservas (más del 85%) se encuentran en el llamado «triángulo de litio¼: salar de Atacama, en Chile; salar de Uyuni, en Bolivia, y salar del Hombre Muerto, en Argentina. En los últimos años, el empleo de microorganismos en la recuperación de metales se ha visto incrementado; sin embargo, hay muy poca información sobre la recuperación de Li por esta vía. En este estudio se trabajó con Rhodococcus sp. A5wh, cepa aislada de Laguna Azul. Los ensayos revelaron que este microorganismo fue capaz de acumular Li (39,52% de Li/g de biomasa en 180 min) y de crecer en presencia de este metaloide hasta una concentración de 1 M. Para comprender los mecanismos implicados en la tolerancia al Li, se llevó a cabo el análisis proteómico comparativo de esta cepa expuesta o no expuesta al Li. Los resultados revelaron 17 spots expresados en forma diferencial. La identificación de las proteínas se realizó porMALDI-TOF/MS. Este estudio constituye el primer enfoque proteómico para dilucidar la estrategia seguida por Rhodococcus en su adaptación al estrés.

Dehalogenase producing bacteria from extreme environment: A review

@#Halogenated compounds create the most important class of xenobiotic which commonly lead to pollution. Some of these compounds are very toxic and cause enormous problems to human health and to the environment. Many of these toxic chemicals have been shown to occur in various extreme habitats. Pollutant-degrading microorganisms, adapted to grow in various environments, play an important role in the biological treatment of polluted extreme habitats. The presence of dehalogenase producing microorganisms in extreme habitat in particular is necessary since the enzyme can catalyze the removal of a halogen atom from a substrate. Therefore, it can reduce the toxicity of the halogenated compound and some are of interest for study in industrial application. Thermophiles, psychrophiles, acidophiles, alkaliphiles and halophiles are types of extremophiles. Knowledge of the biodegradation of toxic chemicals in extreme environment is limited. Here, examples of dehalogenase producing bacteria isolated from various extreme conditions and its special characteristics/features will be discussed in this review.

Putative 3-nitrotyrosine detoxifying genes identified in the yeast Debaryomyces hansenii: in silico search of regulatory sequences responsive to salt and nitrogen stress

Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.

Proteobacteria, extremophiles and unassigned species dominate in a tape-like showerhead biofilm

Abstract The development of showerhead biofilms exposes the user to repeated contact with potentially pathogenic microbes, yet we know relatively little about the content of these aggregates. The aim of the present study was to examine the microbial content of tape-like films found protruding from a domestic showerhead. Culturing showed that the films were dominated by aerobic α- and β-proteobacteria. Three isolates made up almost the entire plate count. These were a Brevundimonas species, a metalophilic Cupriavidus species and a thermophile, Geobacillus species. Furthermore, it was shown that the Cupriavidus isolate alone had a high capacity for biofilm formation and thus might be the initiator of biofilm production. A clone library revealed the same general composition. However, half of the 70 clones analyzed could not be assigned to a particular bacterial phylum and of these 29 differed from one another by only 1–2 base pairs, indicating a single species. Thus both the culture dependent and culture independent characterizations suggest a simple yet novel composition. The work is important as the biofilm is fundamentally different in form (tape-like) and content from that of all previously reported ones, where variously Mycobacterium, Methylobacterium and Xanthomonas species have dominated, and extremophiles were not reported.

Bioprospecting of plant growth promoting psychrotrophic Bacilli from the cold desert of north western Indian Himalayas.

The plant growth promoting psychrotrophic Bacilli were investigated from different sites in north western Indian Himalayas. A total of 247 morphotypes were obtained from different soil and water samples and were grouped into 43 clusters based on 16S rDNA-RFLP analysis with three restriction endonucleases. Sequencing of representative isolates has revealed that these 43 Bacilli belonged to different species of 11 genera viz., Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus and Virgibacillus. With an aim to develop microbial inoculants that can perform efficiently at low temperatures, all representative isolates were screened for different plant growth promoting traits at low temperatures (5-15°C). Among the strains, variations were observed for production (%) of indole-3-acetic acid (20), ammonia (19), siderophores (11), gibberellic acid (4) and hydrogen cyanide (2); solubilisation (%) of zinc (14), phosphate (13) and potassium (7); 1-aminocyclopropane-1-carboxylate deaminase activity (6%) and biocontrol activity (4%) against Rhizoctonia solani and Macrophomina phaseolina. Among all the strains, Bacillus licheniformis, Bacillus muralis, Desemzia incerta, Paenibacillus tylopili and Sporosarcina globispora were found to be potent candidates to be developed as inoculants as they exhibited multiple PGP traits at low temperature.

Bioprospecting of Cellulase Producing Extremophilic Bacterial Isolates from India.

Aim: To isolate and identify the potential extremophilic cellulase producing strain viz., psychrophiles, halophiles, thermophiles and to compare the Cellulase activity from samples collected from different geographical regions of India. Place and Duration of Study: Bharathiar University, Department of Biotechnology, Molecular Microbiology Lab, Coimbatore, Tamilnadu, India, between January to April 2011. Methodology: Cellulase-producing extremophilic bacteria viz., psychrophiles, halophiles, and thermophiles have been isolated from soil samples. According to morphology and pigmentation, 138 distinct bacteria were isolated and screened for cellulase activity by Gram’s iodine–carboxymethylcellulose plate (CMC) assay. On the basis of the cellulase activity, six potent cellulase-producing isolates from each cluster viz., P14, P36, H6, H13, T2 and T3 were selected for 16S rRNA gene based identification. The strains were optimized for maximum cellulase activity at various temperature and pH range. Results: The phylogenetic relationship revealed that P14 and P36 psychrophilic isolates possessed maximum identity with Bacillus simplex (100%) and Arthrobactercitreus (99%), with a cellulase activity of 14.10± 1.73 and 18.27± 0.71 UmL-1 respectively. Likewise, among halophiles, H6 and H13 were identified as Bacillus subtilis and Bacillus endophyticus (99%), with a cellulase activity of 14.87 ± 0.55 and 16.83 ± 0.44 U mL-1, correspondingly. In thermophiles, T2 and T3 showed close proximity with Bacillus amyloliquefaciens and Bacillus megaterium (99%), with a cellulase activity of 21.53 ± 1.30 and 19.93 ± 0.38 U mL-1 respectively. Conclusion: In the present study, the thermophilic isolates showed promising Cellulase activity compared to psychrophiles and halophiles.

Nanoactivator mediated modifications in thermostable amylase from Bacillus licheniformis.

Gram-positive rod-shaped thermophilic bacteria were isolated using samples collected from terrestrial natural thermal spring located at Unkeshwar (Longitude 78.22 degree East to 78.34 degree East, Latitude 19 degree 34' North to 19 degree 40' North), District Nanded, Maharashtra State, India. The isolates were then cultivated using selective media and identified using culture-dependent techniques. One prominent isolate (UN1) exhibited high temperature stability and remarkable amylase production and was identified as Bacillus licheniformis. Amylase production was carried out in starch media and the enzyme was partially purified and characterized for optimization of pH and temperature. Amylolytic activity of the enzyme was determined. Nanoactivator-mediated modifications were carried out to enhance amylolytic activity of the partially purified amylase. Three-fold increase in catalytic efficiency of amylase was obtained after modification.

Life in (and on) the rocks.

Rocks, apart from being ancient records that enlighten us about the geological history of our planet, are dynamic repositories that support life forms central to the sustenance of our biosphere. It is the latter that our discussion will be largely focused on. Life associated with rocks has been documented as early as 1914 (Diels 1914), but it was in the 1960s that Friedmann and colleagues, with their extensive studies on rock-dwellers in hot and cold desert habitats, gave shape to this modern branch of geobiology. The presence of microscopic algae and bacteria was first demonstrated within exposed rocks from hot desert environments such as the Negev and the Sinai (Friedmann and Galun 1974) by electron microscopy and laboratory cultivation methods. Subsequent studies in ortho-quartzite rocks from the Dry Valley area of Antarctica also showed morphologically similar algae (related to the genus Gloeocapsa) to be colonizing areas ~1.5 mm wide parallel to and ~2 mm beneath the rock surface (Friedmann and Ocampo 1976). The results from the latter caught the attention of the scientific community as NASA had been testing their Voyager mission probes on the apparently lifeless cold deserts of Antarctica with the aim of studying a habitat analogous to Mars. In 2005, Walker and colleagues, through the use of culture-independent molecular methods, discovered the microbial colonization of rocks from the extremely acidic (pH~1) Yellowstone geothermal environment. By employing universal PCR primers that targeted 16S rRNA genes from all three domains of life, the authors were able to retrieve sequences phylogenetically related to extant red alga (Cyanidium sp.), bacteria (α-, β-, γ-Proteobacteria; Actinobacteria; Bacteroidetes and Firmicutes) and archaea (Euryarchaeota and Crenarchaeota). The astoundingly high diversity of microbial life forms present in such extreme habitats captured the imaginations of geo(micro)biologists, astrobiologists and microbial ecologists alike. Astrobiologists imagined that if rock interiors could support the major fraction of life in the harshest of environments on earth, then the same could be applicable to other planets such as Mars, and they sensed that there was a need to extend the scope of extraterrestrial life detection missions beyond the mere analysis of top soils. Microbial ecologists wondered if rock-associated life was ubiquitous in the biogeosphere, and geo(micro)biologists hypothesized that the rock micro-habitat offered life (a) protection from intense solar radiations, temperature and desiccation and (b) a supply of nutrients, moisture and growth surfaces. Today, with two dedicated international scientific journals – Geobiology and Geomicrobiology Journal and an ever growing number of papers dealing with rock-associated microbes in microbiological research journals, the concept of ‘life in/on the rocks’ has become as hardened as the rock itself. What is apparently lacking, however, is an understanding among the general public that a ‘dumblooking’ average rock in their gardens (on this planet and perhaps in the gardens of intelligent beings on other planets) could be home to a dynamic assortment of interesting yet diverse living organisms of inevitably microbial nature.

Subjetividade no reconhecimento da vida no universo / Subjetividad en el reconocimiento de la vida en el universo / Subjectivity in the recognition of life in the universe

A vida é primeiramente reconhecida como tal com base em considerações subjetivas a priori. A riqueza das relações subjetivas complexas da vida conosco é a "bioassinatura" mais fundamental, que nos permite construir estratégias para a busca de vida no universo.

la vida se reconoce como tal según consideraciones subjetivas a priori. La riqueza de las relaciones subjetivas complejas con nosotros es la biofirma más fundamental, que nos permite construir estrategias para la búsqueda de la vida en el universo.

Life is primarily recognized as such based on priori subjective considerations. The richness of complex subjective exchanges with us is the most fundamental biosignature, which allows us to build strategies for the search of life in the universe.

Effect of Alkanes on the Production of a Solvent-stable Extracellular Protease by the Organic Solvent Tolerant Bacterium Bacillus licheniformis YP1 / 微生物学通报

In this paper, the effect of 5% (V/V) n-alkanes (e.g, n-Heptane, n-Octane, n-Decane, n-Dodecanen-Tetradecane and n-Hexadecane) on the growth and protease production of organic-solvent-tolerant- bacte-rium Bacillus licheniformis YP1 was studied. 5%(V/V) n-alkanes had no effect on the stability of YP1 prote-ase. 5% (V/V) n-alkanes had no notable influence on the yield of strain YP1 but dramatically affected theprotease production. The presence of n-Heptane, n-Octane and n-Decane deeply repressed the protease pro-duction; however n-Dodecane, n-Tetradecane and n-Hexadecane enhanced the protease production promi-nencely. The concentration of n-Tetradecane (1%-8%, V/V) had a direct ration with the protease production.The detailed experiments showed that the notable increase of protease activity appeared at the late logarithmof cultivation compared with the blank. The cell shape of YP1 strain remarkably decreased when grown inthe presence of n-Tetradecane. This is the first report about the effect of n-alkanes on the protease productionby the solvent tolerant bacterium.