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In the present study, we studied the distribution of silicate mineral weathering bacteria (SWB) in stressed environments that release potassium from insoluble source of mineral. Out of 972 isolates, 340 isolates were positive and mineral weathering potential ranged from 5.55 to 180.05%. Maximum abundance of SWB occurred 44.71% in saline environment followed by 23.53% in low temperature and 12.35% each in high temperature and moisture deficit. Among isolates, silicate mineral weathering efficiency ranged from 1.9 to 72.8 µg mL-1 available K in liquid medium. The phylogenetic tree of SWB discriminated in three clusters viz. Firmicutes, Proteobacteria and Actinobacteria. This is the first report on SWB in stressed environments and identified 27 genera and 67 species which is not reported earlier. Among them Bacillus was the predominant genera (58.60%) distantly followed by Pseudomonas (6.37%), Staphylococcus (5.10%) and Paenibacillus (4.46%). These bacterial strains could be developed as inoculants for biological replenishment of K in stressed soils. Graphical abstract.

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Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate gene expression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application of environmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has proven to be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance, transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to a biocompatible stress resistance inducer such as potassium phosphite, in plants. Further characterization of these miRNAs and their target genes might help to elucidate the molecular mechanisms underlying KPhi-induced resistance.

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Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, ß-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

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Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L–1) at 20 days of cultivation.(AU)

Lignocelulose é o componente mais abundante do meio ambiente e recurso orgânico renovável no solo. Alguns fungos filamentosos têm desenvolvido a habilidade de degradar e utilizar celulose, hemicelulose e lignina como fonte de energia. O objetivo deste trabalho foi analisar o efeito de três fontes de nitrogênio (sulfato de amônio, nitrato de potássio e farelo de soja) na atividade enzimática de Lentinula edodes EF 50 utilizando como substrato serragem de E. benthamii. Foi aplicado um planejamento experimental de mistura com três repetições no ponto central constituído de sete tratamentos (T) de iguais concentrações em nitrogênio de sulfato de amônia, nitrato de potássio e farinha de soja cozida. Foram determinadas a atividade enzimática da avicelase, carboximetilcelulase, β-glicosidase, xilanases e manganês peroxidase. Foram avaliados o teor de umidade, pH, atividade de água (aw) e análise qualitativa do crescimento micelial em 8 tempos de cultivo. Os resultados mostraram efeito negativo na produção das enzimas nos tratamentos com máxima concentração de sulfato de amônia e nitrato de potássio. Os tratamentos com farinha de soja cozida expressaram maiores atividades enzimáticas, nos tempos de 3, 6 e 9 dias de cultivo exceto na atividade do manganês peroxidase. A maior produção foi observada no tratamento com sulfato de amônia e farinha de soja cozida (83.86 UI.L–1) em 20 dias de cultivo.(AU)

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Abstract Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L–1) at 20 days of cultivation.

Resumo Lignocelulose é o componente mais abundante do meio ambiente e recurso orgânico renovável no solo. Alguns fungos filamentosos têm desenvolvido a habilidade de degradar e utilizar celulose, hemicelulose e lignina como fonte de energia. O objetivo deste trabalho foi analisar o efeito de três fontes de nitrogênio (sulfato de amônio, nitrato de potássio e farelo de soja) na atividade enzimática de Lentinula edodes EF 50 utilizando como substrato serragem de E. benthamii. Foi aplicado um planejamento experimental de mistura com três repetições no ponto central constituído de sete tratamentos (T) de iguais concentrações em nitrogênio de sulfato de amônia, nitrato de potássio e farinha de soja cozida. Foram determinadas a atividade enzimática da avicelase, carboximetilcelulase, β-glicosidase, xilanases e manganês peroxidase. Foram avaliados o teor de umidade, pH, atividade de água (aw) e análise qualitativa do crescimento micelial em 8 tempos de cultivo. Os resultados mostraram efeito negativo na produção das enzimas nos tratamentos com máxima concentração de sulfato de amônia e nitrato de potássio. Os tratamentos com farinha de soja cozida expressaram maiores atividades enzimáticas, nos tempos de 3, 6 e 9 dias de cultivo exceto na atividade do manganês peroxidase. A maior produção foi observada no tratamento com sulfato de amônia e farinha de soja cozida (83.86 UI.L–1) em 20 dias de cultivo.

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This study aimed to evaluate under field conditions the efficiency in the use ofN coated with urease inhibitor in maize. The experiment was conducted in the year of 2007/2008. The experimental design was a randomized block design in a factorial 2 x 6, with five repetitions, constituted the N sources (common and coated with urease inhibitor) and levels (0, 40, 80, 120, 160 and 200 kg ha-1 of N) sidedressing nitrogen application in the growth stage V4. Based on the data obtained were determined recovery efficiencies, utilization, agronomic and physiological N applied. In all cases, the efficiency levels for maize were influenced by levels of sidedressing nitrogen application, in which increasing levels of N resulted in a decrease of the efficiencies, regardless of the source being common urea or coated with urease inhibitor.

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Urea has been considered as a promising alternative nitrogen source for the cultivation of Arthrospira platensis if it is possible to avoid ammonia toxicity; however, this procedure can lead to periods of nitrogen shortage. This study shows that the addition of potassium nitrate, which acts as a nitrogen reservoir, to cultivations carried out with urea in a fed-batch process can increase the maximum cell concentration (X(m) ) and also cell productivity (P(X) ). Using response surface methodology, the model indicates that the estimated optimum X(m) can be achieved with 17.3 mM potassium nitrate and 8.9 mM urea. Under this condition an X(m) of 6077 ± 199 mg/L and a P(X) of 341.5 ± 19.1 mg L(-1) day(-1) were obtained.

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The kinetic properties of a microsomal gill (Na(+), K(+)) ATPase from the blue crab, Callinectes danae, acclimated to 15 per thousand salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na(+), K(+))-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V=102.9+/-4.3 U.mg(-1) with K(0.5)=1.7+/-0.1 mmol.L(-1), while stimulation by magnesium (V=93.7+/-2.3 U.mg(-1); K(0.5)=1.40+/-0.03 mmol.L(-1)) and potassium ions (V=94.9+/-3.5 U.mg(-1); K(0.5)=2.9+/-0.1 mmol.L(-1)) was cooperative. K(+)-phosphatase activity was also stimulated by ammonium ions to a rate of V=106.2+/-2.2 U. mg(-1) with K(0.5)=9.8+/-0.2 mmol.L(-1), following cooperative kinetics (n(H)=2.9). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4) (+) ions. Sodium ions (K(I)=22.7+/-1.7 mmol.L(-1)), and orthovanadate (K(I)=28.1+/-1.4 nmol.L(-1)) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K(I)=142.0+/-7.1 micromol.L(-1)). Western blotting analysis revealed increased expression of the (Na(+), K(+))-ATPase alpha-subunit in crabs acclimated to 15 per thousand salinity compared to those acclimated to 33 per thousand salinity. The increase in (Na(+), K(+))-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na(+), K( (+) ))-ATPase alpha-subunit; phosphate-hydrolyzing enzymes other than (Na(+), K(+))-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.

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The cyanobacterium Spirulina platensis was cultivated in bench-scale miniponds on bicarbonate/carbonate solutions using urea as nitrogen source. To minimize limitation and inhibition phenomena, urea was supplied semicontinuously using exponentially increasing feeding rates. The average growth rates obtained alternately varying the total mass of urea added per unit reactor volume (275 < mT < 725 mg/L) and the total feeding time (9 < tT < 15 d) clearly evidenced nitrogen limitation for mT< 500 mg/L and excess nitrogen inhibition above this threshold. The time behavior of the specific growth rate at variable urea feeding patterns allowed estimation of the time-dependent Gibbs energy dissipation for cell growth under the actual depletion conditions of fed-batch cultivations. Comparison of the yield of growth on Gibbs energy obtained using either urea or KNO3 pointed to the preference of S. platensis for the former nitrogen source, likely owing to more favorable bioenergetic conditions.

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AIMS: The aim of this study was to apply a group specific PCR system followed by denaturing gradient gel electrophoresis (DGGE) analysis to evaluate the effect of oil contamination and the biostimulation process on the diversity of Pseudomonas populations in soil ecosystems. METHODS AND RESULTS: Direct DNA extraction from biostimulated- and oil-contaminated soil samples was performed. Primers specific for the genus Pseudomonas spp. were used to amplify 16S rRNA genes and then a semi-nested PCR reaction was applied to obtain smaller fragments for comparing the PCR products by DGGE. Whether in bulk, oil-contaminated or biostimulated soils, the DGGE profiles revealed little change in Pseudomonas community throughout the 270 days of experiment. The presence of a few additional bands observed only in treated samples indicated that a bacterial shift occurred with the addition of nutrients and with oil contamination. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of semi-nested PCR and DGGE was found to be a rapid and sensitive technique to study the diversity within the genus Pseudomonas and may be suitable for further studies concerning the role of this bacterial group in large-scale oil-contaminated areas.

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Candida lipolytica IA 1055 produced extracellular biosurfactants with emulsification activity by fermentation using babassu oil and D-glucose as carbon sources. Natural seawater diluted at 50 percent supplemented with urea, ammonium sulfate, and phosphate was used as economic basal medium. The best results were achieved with the YSW-B2 medium, which contained urea, ammonium sulfate, and babassu oil and with YSW-B3 medium, which contained urea, ammonium sulfate, phosphate, and babassu oil, kept under fed batch fermentation for 60 hours with 5 percent of babassu oil. For the two media, the maximum specific growth rates were 0.02 h-1 and 0.04 h-1; the generation times were 34.6 h-1 and 17.3 h-1, and the emulsification activities were 0.666 and 0.158 units, respectively. The molecules of these new bioemulsifiers were contituted of carbohydrates, proteins and lipids.

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We evaluated the interaction between ascorbic acid (AA) and (+)-catechin (CTCH) in potassium phosphate solution, pH 7.4 (PPS) and in human plasma. In both systems, the oxidation was started by adding 2,2'-azobis-(2-amidinopropane) clorhidrate (AAPH). The concentrations of AA and CTCH were determined by HPLC using electrochemical detection. In PPS, CTCH was oxidized by AAPH (50 mM), in either the absence or presence of different initial concentrations of AA (25-200 microM). In the presence of AA, CTCH depletion was delayed, an effect that was dependent upon the initial concentration of AA. When 100 microM AA was added after the oxidation had begun, CTCH depletion was arrested for 30 min. The kinetics of AA oxidation by AAPH was also characterized in PPS. AA (100 microM) was completely consumed after 60 min of reaction at 37 degrees C, in both the absence and presence of 100 mM CTCH. When human plasma was incubated with 50 mM AAPH in the absence of added CTCH, AA was completely consumed after 45-60 min. CTCH did not prevent AA depletion in human plasma at the concentrations tested (10, 50 100 microM). The results point out that AA is able to protect other aqueous soluble antioxidants, e.g.: CTCH.

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We evaluated the interaction between ascorbic acid (AA) and (+)-catechin (CTCH) in potassium phosphate solution, pH 7.4 (PPS) and in human plasma. In both systems, the oxidation was started by adding 2,2'-azobis-(2-amidinopropane) clorhidrate (AAPH). The concentrations of AA and CTCH were determined by HPLC using electrochemical detection. In PPS, CTCH was oxidized by AAPH (50 mM), in either the absence or presence of different initial concentrations of AA (25-200 microM). In the presence of AA, CTCH depletion was delayed, an effect that was dependent upon the initial concentration of AA. When 100 microM AA was added after the oxidation had begun, CTCH depletion was arrested for 30 min. The kinetics of AA oxidation by AAPH was also characterized in PPS. AA (100 microM) was completely consumed after 60 min of reaction at 37 degrees C, in both the absence and presence of 100 mM CTCH. When human plasma was incubated with 50 mM AAPH in the absence of added CTCH, AA was completely consumed after 45-60 min. CTCH did not prevent AA depletion in human plasma at the concentrations tested (10, 50 100 microM). The results point out that AA is able to protect other aqueous soluble antioxidants, e.g.: CTCH.

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We report kinetic data of penicillin hydrolysis catalyzed by beta-lactamase entrapped in reverse micelles formed with cetyl trimethylammonium bromide (CTAB), n-octane, hexanol and aqueous buffer. The K(cat) of this diffusion-limited reaction can be improved in aqueous buffer by a factor of 1.1-1.2 just by increasing the phosphate buffer concentration from 50 to 100 mM. In reverse micelles, increasing the buffer concentration has little effect on K(cat) when the size of the empty micelle is below the size of the protein. However, in larger micelles, the effect is enhanced and the K(cat) improves several fold, changing the form of the curve of K(cat) versus Wo from bell-shaped to almost hyperbolic. The results indicate that micellar exchange and internal diffusion may limit the reaction in reverse micelles and provide further evidence that the form of the curve depends on other factors besides the relationship between the size of the enzyme and that of the empty reverse micelle.

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Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate. One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms. A Tn5-20 mutation of this glnB was Nif negative.

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The effect of ergosterol depletion by ketoconazole on the leishmanicidal activity of the pore-forming antibiotic amphotericin B (AmB) was investigated. Leishmania mexicana promastigotes were lysed within minutes by the addition of micromolar concentrations of AmB (0.5 microM) but became insensitive to AmB after growth in the presence of ketoconazole (0.25 microM, 90 h). Lipid chromatographic analysis indicated that under such conditions, ketoconazole depleted the major Leishmania sterols, dehydroepisterol and ergosterol. Plasma membrane vesicles prepared from ketoconazole-treated promastigotes exhibited a much reduced enhancement of their salt permeability after the addition of AmB at concentrations as high as 5 microM. This finding clearly indicates that upon ketoconazole treatment, the capacity of pore formation by the antibiotic is substantially impaired. The reduction of desmethyl sterols by ketoconazole was accompanied by a significant increase of 14-alpha-methyl sterols, but exogenous cholesterol remained unchanged. This ability of Leishmania promastigotes to incorporate cholesterol from the external medium may explain why ketoconazole-treated cells exhibited a much decreased but significative response to AmB when they were exposed to high AmB concentrations (2.5 or 5.0 microM). Parallel measurements by using a fluorescence energy transfer method indicated that binding of AmB to ketoconazole-treated Leishmania promastigotes and heat-transformed leishmanias was also decreased but to different extents, a finding that may be related to the differences in their sterol content. The results obtained clearly indicate that the specific interaction of AmB with desmethyl sterols, such as dehydroepisterol, ergosterol, and even exogenous cholesterol, is an absolute requirement for the lethal action exerted by this polyene antibiotic on L. mexicana promastigotes.

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