RESUMO
Objetivou-se avaliar aspectos físico-químicos e microbiológicos do farelo úmido de glúten de milho (FUGM) em função do tempo de ensilagem. O FUGM foi avaliado em silos experimentais e, nos dias um, três, sete, 14, 21, 28 e 42, foram realizadas as avaliações físicas, químicas e microbiológicas da silagem. O delineamento experimental foi inteiramente casualizado com quatro repetições e obtidas equações de regressão em função do tempo. O tempo de ensilagem não afetou (P>0,05) o percentual de fibra em detergente neutro, fibra em detergente ácido, proteína bruta, matéria mineral, extrativos não-nitrogenados, nutrientes digestíveis totais e contagem de clostrídios. Os teores de matéria seca, extrato etéreo, perdas por efluentes, perdas por gases e população de bactérias láticas variaram entre 46,54 e 49,05%; 0,96 e 1,71%; 17,97 e 35,14g kg-1; 1,90 e 5,68% e 5,0 e 6,9log UFCg-1 de silagem, respectivamente, todos com equações com r2≤0,6.O pH (Y=4,12926-0,00894dias+0,00024126dias2,r2=0,75) e a temperatura (Y=19,82657+0,11734dias-0,00502dias2,r2=0,73) tiveram seus menores e maiores valores, respectivamente, em 18,5 e 11,7 dias. A contagem de bolores e leveduras sofreu redução em função do tempo de ensilagem (Y=6,8983-0,0363dias,r2=0,84). A ensilagem permite eficiente conservação dos aspectos químicos, microbiológicos e físicos do FUGM até 42 dias de ensilagem.
This study aimed to evaluate physicochemical and microbiological aspects of wet corn gluten feed (WCGF) according to time of ensilage. The WCGF was evaluated in experimental silos and after one, three, seven, 14, 21, 28 and 42 days, physical, chemical and microbiological evaluation of the silage was carried out. The results were analyzed as a completely randomized design with four replications and regression equations as a function of time. The number of days of ensiling did not affect neutral detergent fiber, acid detergent fiber, crude protein, mineral matter, non-nitrogenous extract and total digestive nutrients percentages, as well as clostridia count. Dry matter, ether extract, effluents losses and percentages of gas losses and lactic acid bacteria count with variations of 46.54 and 49.05%; 0.96 and 1.71%; 17.97 and 35.14g kg-1; 1.90 and 5.68% and 5.0 and 6.9log CFUg-1 of silage, respectively, all had low coefficient of determination (r2≤0.6).The pH (Y=4.12926-0.00894days+0.00024126days2,r2=0.75) and temperature (19.82657+0.11734days-0.00502days2,r2=0.73) had their minor and major values, respectively, in 18.5 and 11.7 days. Moulds and yeasts count decrease with time of ensilage (Y=6.8983-0.0363days,r2=0.84). The ensiling allows efficient conservation of the chemical, microbiological and physical aspects of WCGF until 42 days of ensilage.
RESUMO
We designed a strategy for the sequencing and bioinformatical characterization of the 1,3-propanediol operon regulator genes from the Colombian Clostridium sp. strain IBUN13A, which is taxonomically related to Clostridium butyricum. Three genes are proposed to be involved in the operon's transcriptional activity, the dhaS and dhaA genes through a two-component system and the third gene named dhaY, which encodes a putative transcriptional regulator similar to the domains of the dhaS/A system. Phylogenetic analyses indicated that the predicted proteins had a modular structure consisting of domains homologous to different signal transduction systems, but had significant differences concerning their conserved residues, pointing to the possibility that they constitute ancestral domains. In accordance with the prediction of functions, we propose a mechanism of regulation of the proteins studied of the 1,3-propanediol operon of the native strain, as a response to the presence of glycerol in the medium, which provides valuable information on the overall regulation of the glycerol metabolism in Clostridium sp.
Se diseñó una estrategia de amplificación, secuenciación y caracterización bioinformática de los genes reguladores del operón 1,3-propanediol (1,3-PD) de la cepa nativa colombiana Clostridium sp. IBUN 13A, relacionada taxonómicamente con Clostridium butyricum. Se identificaron tres genes que pueden estar involucrados en la regulación transcripcional de dicho operón: los genes dhaS y dhaA -a través de un sistema de transducción de señales de dos componentes-y un tercer gen que se denominó dhaY, que codifica para un regulador transcripcional putativo, similar a los dominios presentes en las proteínas del sistema DhaS/A. Los análisis filogenéticos indican que las proteínas predichas presentan una estructura modular con dominios homólogos a diferentes sistemas de transducción de señales, pero muestran diferencias importantes en los residuos conservados, lo que sugiere que podrían ser estos los dominios ancestrales. La predicción de funciones postula un mecanismo de regulación de las proteínas estudiadas sobre el promotor del operón 1,3-PD de la cepa nativa como respuesta a la presencia de glicerol en el medio, lo cual aporta información importante sobre la regulación global del metabolismo del glicerol en Clostridium sp.
Nesta pesquisa foi feita uma estratégia para a amplificacäo, sequenciamento e caracterizacäo bioinformática dos genes reguladores do operon 1,3 propanodiol (1,3-PD) da cepa colombiana Clostridium sp. IBUN 13A, relacionada taxonomicamente com o Clostridium butyricum. Tèm sido identificados tres genes que podem estar envolvidos na regulacáo transcricional do operon. Os genes dhaS e dhaA por meio de um sistema de dois componentes e o terceiro gene nomeado de dhaY, que codifica para um regulador transcridonal putativo, parecido com os dominios presentes nas proteínas do sistema DhaS/A. A análise filogenètica mostra que estas proteínas apresentam uma estrutura modular com dominios homólogos a diferentes sistemas de traducáo de sinais, mas pressupöem diferencas importantes nos residuos conservados, indicando provavelmente que possam constituir os dominios ancestrais. De acordo com a predicáo de funcöes, é postulado um mecanismo de regulacáo do sistema DhaS/A, DhaY sobre o promotor do operon 1,3-DP da cepa nativa, como resposta à presenca de glicerol no meio, aportando informacöes importantes da regulacáo global do metabolismo do glicerol no Clostridium sp.
RESUMO
El objetivo de este estudio fue analizar las rutas metabólicas para la producción de solventes y degradación de celulosa en cepas colombianas promisorias del género Clostridium. Para ello se diseñaron sondas de hibridación que sirvieran para posteriores estudios de mejoramiento genético de las cepas. Se construyó la base de datos denominada MULTICLOST en Microsoft Access® con las secuencias de 485 genes involucrados en las rutas metabólicas arriba mencionadas, provenientes de 45 especies bacterianas y 10 especies fúngicas. Los genes fueron agrupados de acuerdo al tipo de enzima y a los dominios catalíticos o de unión a sustrato en el caso de las celulasas. Cada grupo se sometió a alineamiento múltiple en ClustalW 1.83 y con base en los resultados se crearon subgrupos de similitud mayor al 50%. Se localizaron secuencias conservadas de longitud mayor a 19 nucleótidos en GeneDoc 2.6.002 y sus valores termodinámicos fueron estimados con GeneRunner v3.05, mientras que la sensibilidad y especificidad fue verificada por búsquedas en GenBank usando BLASTN 2.2.8. En total se obtuvieron 94 secuencias conservadas con las siguientes características: longitud promedio de 24 nucleótidos, Tm promedio de 65,8 ºC y contenido de (G+C) entre 14,3 y 60,0%. Se determinó que ninguna de las sondas diseñadas forma estructuras secundarias estables con Tm superior a 36,1 ºC. De acuerdo a sus características y valores termodinámicos, todas las sondas podrían ser utilizadas en la construcción de un microarreglo o en reacciones de PCR para la identificación de regiones relevantes en el mejoramiento del proceso por ingeniería metabólica.
The goal of the present study was to analyze the metabolic pathways involved in solvent production and cellulose consumption by promising Colombian native strains of the genus Clostridium. Therefore a set of oligonucleotide probes was designed, with the aim of analyzing potential targets for genetic improvement of the Colombian strains. The database named MULTICLOST was created in Microsoft Access® using the sequences from 485 genes involved in solventogenesis, 1,3propanodiol production and cellulolysis from 45 bacterial and 10 fungal species. The genes were grouped according to their respective enzyme function and to the catalytic domain or the substrate binding domain in the case of cellulases. ClustalW 1.83 was used for multiple alignment of every group. Subgroups of sequences with more than 50% identity among themselves were created. Conserved sequences longer than 19 nucleotides were identified using GeneDoc 2.6.002 and their thermodynamic values were calculated with GeneRunner v3.05, while their sensitivity and specificity were verified by searching in GenBank with BLASTN 2.2.8. Ninetyfour conserved sequences were obtained with an average 24nucleotide length, 65.8ºC average Tm and a (G+C) content between 14.3% and 60.0%. None of these probes forms stable secondary structures at temperatures higher than 36.1ºC. According to the former results, all of the probes could be used in an oligonucleotide microarray or in PCR reactions for the identification of metabolic targets for improvement of the industrial process.
RESUMO
A anaerobic hydrogen-producing strain HR-1 was isolated from compost. Phylogenetic analysis based on 16S rRNA sequence similarity indicates that strain HR-1 is the closest relative to Clostridium ace- tobutylicum ATCC 824, with the similarity of 96%. Biological characteristics and phylogenetic analysis of 16S rRNA gene indicate that HR-1 is a new species named Clostridium sp. HR-1. Cells are Gram-positive, mobile rod-shaped. Spores and flagellums were no observed. Temperature range for growth is 10?C to 45?C (optimum temperature 37?C~39?C), and range pH for growth is 4.0 to 10.0 (optimum pH 7.5~8.0). H2, CO2, acetate, butyrate and a little ethanol are the end products of PYG fermentation. Strain HR-1 has the ability to use organic nitrogen and inorganic nitrogen sources for growth and hydrogen production, and yeast extract is the optimum nitrogen source for hydrogen production. Strain HR-1 produces hydrogen from xylose (3 g/L) at 37?C and initial pH 6.5, the hydrogen yields and maximal hydrogen production rate are 1.84 mol-H2/mol-xylose and 10.52 mmol-H2/h?g-cdw, respectively. Strain HR-1 is able to utilize glucose, galactose, fructose, mannose and cellobiose for hydrogen production and the hydrogen yields from glucose is 2.36 mol-H2/mol-glucose.