Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens

Background Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ( ${\mathrm{NO}}_{3}^{-}$ NO 3 − ) to nitrite ( ${\mathrm{NO}}_{2}^{-}$ NO 2 − ). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. Methods Series of oxic and anoxic cultures of strain JAM1 were performed with N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − or sodium nitroprusside, and growth and N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − , ${\mathrm{NO}}_{2}^{-}$ NO 2 − and N2 concentrations were measured. Ammonium ( ${\mathrm{NH}}_{4}^{+}$ NH 4 + )-free cultures were also tested to assess the dynamics of N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − and ${\mathrm{NO}}_{2}^{-}$ NO 2 − . Isotopic labeling of N2O was performed in 15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2 double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc-type nitric oxide reductase (cnorB1 and cnorB2) and nitrous oxide reductase (nosZ), and also nnrS and norR that encode NO-sensitive regulators. Results Strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory ${\mathrm{NO}}_{2}^{-}$ NO 2 − reductase, ${\mathrm{NO}}_{3}^{-}$ NO 3 − -amended cultures produce N2O, representing up to 6% of the N-input. ${\mathrm{NO}}_{2}^{-}$ NO 2 − was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1, cnorB2, nnrS and norR during the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures. Discussion By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using ${\mathrm{NO}}_{3}^{-}$ NO 3 − and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities

Separation and characterization biochemistry of the enzyme 1,3-propanediol oxidoreductase coming from a native strain of Clostridium spp IBUN 158

El propósito de este trabajo fue purificar y caracterizar la enzima 1,3-propanodiol doxidoreductasa (1.1.1.202) perteneciente a la vía reductiva de la ruta de glicerol, procedente de la cepa nativa de Clostriduum sp IBUN 158b. Esta enzima es capaz de reducir 3-Hidroxipropionaldehido y trasformalo a 1,3- propanodiol, con la producción simultanea de NAD+. En este estudio la enzima fue obtenida a partir de la producción de extracto protéico crudo, realizando bajo la técnica de sonicación y en la cual se estableció que 12 ciclos de 30 segundos eran los necesarios para obtener muestras homogéneas con concentraciones de proteina que iban de 2.6 a 3.1 mg/ml. La enzima fue purificada empleando la técnica de cromatografía por intercambio aniónico con porcentajes de recuperación que alcanzaban el 68%, actividades enzimáticas de 0.0094 μmol/min y actividades específicas que se encontraban alrededor de 0.067 μmol/min/mg. Se analizaron diferentes parámetros como temperatura, pH, parámetros cinéticos Km y Vmax, efecto de iones divalentes, masa molecular y pl teórico para evaluar la influencia que ejercen sobre la actividad de la enzima. Los resultados establecidos para la 1,3-propanodiol deshidrogenasa fueron una temperatura de 30°C, un pH de 10.0, un comportamiento alostérico y se pudo establecer que la concentración de 1,3-PD 100 mM y de NAD+ 2 rnIV1 aumentaban la actividad de la enzima hasta en un 100%; una concentración de iones de MnCl2 1mM que logro aumentar la actividad en un 50%, una masa molecular de 71 +/- 3 KDa y un pl teórico de 5.9. / Abstract. The purpose of this study was to purify and characterize the enzyme 1,3-propanediol oxidoreductase (1.1.1.202) belonging to the reductive way of the route of glycerol from the native strain of Clostridium sp 1BUN 158B. This enzyme is capable of reducing 3-Hidroxipropionaldehido and transform to 1,3-propanediol, with the simultaneous production of NAD +. In this study the enzyme was obtained from the production of a crude protein extract, carried out under sonication technique and in which it was established that 12 cycles of 30 seconds were needed to obtain homogeneous samples with protein concentrations ranging from 2.6 to 3.1 mg/ml. The enzyme was purified using the technique of Anionic-exchange chromatography with a recovery that reached 68% enzymatic activity of 0.0094 pmol/min and specific activities were approximately 0.067 pmol/min/mg. We analyzed different parameters such as temperature, pH, kinetic parameters Km and Vmax, the effect of divalent ions, molecular mass and theoretical pl to assess the influence on the activity of the enzyme. The results established for the 1,3-propanediol dehydrogenase were a temperature of 30° C, pH 10.0, allosteric behavior and it was found that the concentration of 1,3-PD 100 mM and 2 mM NAD + increased the activity of the enzyme up to 100%, a concentration of 1 mM MnC|2 ions to achieve enhanced activity by 50%, a molecular mass of 71 + / - 3 kDa and a theoretical pl of 5.9.Master scientae en microbiologíaMaestrí

Co-culturing Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 allows sustainable denitrifying activities under marine conditions

Background Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities of a methanol-fed, fluidized-bed marine denitrification system. Strain NL23 possesses the complete denitrification pathway, but cannot grow under marine conditions in pure cultures. Strain JAM1 is a marine bacterium that lacks genes encoding a dissimilatory nitrite (NO2−) reductase and therefore cannot reduce NO2−. Here, we report the characterization of some of their physiological traits that could influence their co-habitation. We also perform co-cultures to assess the potential synergy between the two strains under marine and denitrifying conditions. Methodology Anoxic planktonic pure cultures of both strains were grown with different concentrations of nitrate (NO3−). Anoxic planktonic co-cultures could only be cultured on low NaCl concentrations for strain NL23 to grow. Biofilm co-cultures were achieved in a 500-mL bioreactor, and operated under denitrifying conditions with increasing concentrations of NaCl. NO3− and NO2− concentrations and the protein content were measured to derive the denitrification rates. The concentrations of both strains in co-cultures were determined by quantitative PCR (qPCR). Ectoine concentration was measured by mass spectrometry in the biofilm co-culture. The biofilm was visualized by fluorescence in situ hybridization. Reverse-transcription-qPCR and RNA-seq approaches were used to assess changes in the expression profiles of genes involved in the nitrogen pathways in the biofilm cultures. Results Planktonic pure cultures of strain JAM1 had a readiness to reduce NO3− with no lag phase for growth in contrast to pure cultures of strain NL23, which had a 2-3 days lag phase before NO3− starts to be consumed and growth to occur. Compared to strain NL23, strain JAM1 has a higher µmax for growth and higher specific NO3− reduction rates. Denitrification rates were twice higher in the planktonic co-cultures than those measured in strain NL23 pure cultures. The biofilm co-cultures showed sustained denitrifying activities and surface colonization by both strains under marine conditions. Increase in ectoine concentrations was observed in the biofilm co-culture with the increase of NaCl concentrations. Changes in the relative transcript levels were observed in the biofilm culture with genes encoding NapA and NapGH in strain NL23. The type of medium had a great impact on the expression of genes involved in the N-assimilation pathways in both strains. Conclusions These results illustrate the capacity of both strains to act together in performing sustainable denitrifying activities under marine conditions. Although strain JAM1 did not contribute in better specific denitrifying activities in the biofilm co-cultures, its presence helped strain NL23 to acclimate to medium with NaCl concentrations >1.0%

Proponer una aproximación metodológica para el estudio sobre las tendencias de consumo en Bogotá

Para identificar las motivaciones que llevan al ser humano a consumir los diferentes productos y servicios que se encuentran en el mercado, es necesario nombrar los conceptos básicos que se relacionan en las tendencias de consumo, así como identificar las empresas que a nivel internacional y nacional específicamente en la ciudad de Bogotá se dedican a este tipo de estudios., conociendo el enfoque que cada una le da en su proceso de investigación. Después decidimos resumir este tipo de información en una matriz que nos permite conocer de una manera más fácil: público objetivo, Resultados, Periodicidad, Metodología utilizada y resultados de las empresas seleccionadas, Para así proceder con el planteamiento de nuestra metodología teniendo en cuenta las variables que vamos a utilizar y finalmente realizar una prueba piloto con el instrumento seleccionado sacando de este conclusiones y recomendaciones que se deben tener en cuenta en el momento que se desee aplicar dicha metodología a una muestra real.INTRODUCCION OBJETIVOS 1 MARCO CONCEPTUAL 11 Definiciones de Consumo 111 Consumo desde el área económica 112 Consumo desde el área de mercadeo 113 Consumo desde la Psicología del Consumidor 12 Definiciones de Consumidor 121 Consumidor desde el Área Económica 122 Consumidor desde el Área de Mercadeo 123 Psicología Del Consumidor 13 Definiciones de Tendencias de Consumo 131 Tendencia 132 Tendencia de Consumo desde el Área de Mercadeo 133 Tendencia de Consumo desde el Área de Economía 2 ORGANIZACIONES QUE ESTUDIAN LAS TENDENCIAS DE CONSUMO A NIVEL INTERNACIONAL 21 Millward Brown METODOLOGÍA PARA TENDENCIAS DE CONSUMO EN BOGOTÁ 22 AC Nielsen Company 23 Append Investigación de Mercados 24 Latin American Markets 25 Collect GFK 26 Bain & Company 27 Deloitte México 28 American Martketing Asociation 29 Popcorn Marketing 210 Mckinssey And Company 211 Trendwatchingcom 3 ORGANIZACIONES QUE ESTUDIAN LAS TENDENCIAS DE CONSUMO A NIVEL NACIONAL ESPECIFICAMENTE EN LA CIUDAD DE BOGOTÁ 31 Cámara de Comercio de Bogotá 4 EJEMPLO DE TENDENCIA DE CONSUMO INTERNACIONAL Y NACIONAL 41 Trendwatching 42 Once Tendencias de Consumo por Daniel Naranjo en Bogotá 5 DESARROLLO DE LA METODOLOGÍA 51 Variables escogidas para la propuesta metodológica 511 Investigación Documental o de Gabinete (Desk Research) - Análisis de Fuentes Secundarias 5111 Estudios Ómnibus 52 Desarrollo del Manual 521 Paso No 1 522 Paso No 2 METODOLOGÍA PARA TENDENCIAS DE CONSUMO EN BOGOTÁ 523 Paso No 3 524 Paso No 4 525 Paso No 5 526 Paso No 6 527 Paso No 7 6 PRUEBA PILOTO 61 Fuentes Primarias 62 Fuentes Secundarias 7 RESULTADOS DE LA PRUEBA PILOTO 71 Estudio Ómnibus 711 Observaciones de la prueba 712 Observaciones del instrumento 72 Fuentes Secundarias 721 Observaciones 73 Observación Fotográfica 731 Observaciones CONCLUSIONES RECOMENDACIONES GLOSARIO REFERENCIAS ANEXOSPregradoMercadeo y PublicidadMercadeo y Publicida

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1

Background Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ( ${\mathrm{NO}}_{3}^{-}$ NO 3 − ) to nitrite ( ${\mathrm{NO}}_{2}^{-}$ NO 2 − ). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. Methods Series of oxic and anoxic cultures of strain JAM1 were performed with N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − or sodium nitroprusside, and growth and N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − , ${\mathrm{NO}}_{2}^{-}$ NO 2 − and N2 concentrations were measured. Ammonium ( ${\mathrm{NH}}_{4}^{+}$ NH 4 + )-free cultures were also tested to assess the dynamics of N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − and ${\mathrm{NO}}_{2}^{-}$ NO 2 − . Isotopic labeling of N2O was performed in 15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2 double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc-type nitric oxide reductase (cnorB1 and cnorB2) and nitrous oxide reductase (nosZ), and also nnrS and norR that encode NO-sensitive regulators. Results Strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory ${\mathrm{NO}}_{2}^{-}$ NO 2 − reductase, ${\mathrm{NO}}_{3}^{-}$ NO 3 − -amended cultures produce N2O, representing up to 6% of the N-input. ${\mathrm{NO}}_{2}^{-}$ NO 2 − was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1, cnorB2, nnrS and norR during the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures. Discussion By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using ${\mathrm{NO}}_{3}^{-}$ NO 3 − and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities