Stability assessment and improvement of enzymatic activity of the endoglucanases from bacillus sp. Ar03

The lignocellulosic biomass is well known like a promising source to biorefinery due to its abundant and its renewable feature. Cellulose, the major compound of this material, needs the cooperative action of at least three types of enzymes to be degraded: exoglucanases, endoglucanases and β-glucosidases. Microorganisms and their enzymes are biotechnical tools that nature has designed to utilize biomass that is present in the habitat around them. In this sense, Bacteria are extensively considered as a source of novel cellulases because of their diversity and due to their higher growth rate and their extensive repertoire of glycoside hydrolases. The aim of the present work was to produce and to characterize endoglucanases from Bacillus sp. AR03, isolated from sugarcane bagasse liquor, to further generate lignocellulosic hydrolysates. The isolate AR03 was grown in a peptone broth amended with carboxymethyl cellulose 1% (w/v) and sucrose 1%(w/v) at 30 °C and 200 rpm. After 48 h, the culture supernatant was recovered by centrifugation and the endoglucanase activity was estimated by measuring reducing sugar released from CMC by the dinitrosalicylic acid (DNS) method. Zymograms of the culture supernatant were carried out by native PAGE. The effects of temperature, pH, cations and others additives such as EDTA, PEG, SDS and Tween 80 were assayed to assess their influence on the activity and stability of the endoglucanases produced. The enzyme production reached 3 IU/mL in the crude extract (culture supernatant) and the optimal endoglucanase activity was registered at 60 °C and pH 6.0. The evaluated enzymatic extracts showed that the enzyme activity was completely retained after pretreatments at temperatures £ to 40 °C, although it did not show thermal stability after preheating at 60 °C for one hour. Endoglucanases from AR03 isolate maintained approximately 80% of the total activity within a wide range of pH (3.0 to 10.0). The native PAGE revealed at least three bands with endoglucanase activity, having apparent molecular masses of 286, 208 and 157 kDa. Even when most of the effectors assayed did not affect significantly the enzymatic activity, the addition of Mn2+ and Co2+ (5 mM) to the enzymatic reaction mixture produced a noteworthy improvement of the endoglucanase activity from the crude extracts. The endoglucanase activity was upgraded as much as 150% and 80% when salts containing Mn2+ and Co2+ were added, respectively. Those increments were confirmed by means of HPLC measurements since it has been reported interference between some divalent cations and the DNS reagent. The results regarding the broad range of pH stability and the strong improvement of enzymatic activity by the presence of manganese are the most relevant features of the endoglucanases from Bacillus sp. AR03 to be considered as promising for further studies and for biotechnological applications.Fil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Manfredi, Adriana Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Perotti, Nora Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaXI Congreso de Microbiología GeneralCórdobaArgentinaAsociación Civil de Microbiología Genera

Proteomic analysis of secretomes from Bacillus sp. AR03: characterization of enzymatic cocktails active on complex carbohydrates for xylooligosaccharides production

Bacillus sp. AR03 have been described as an important producer of carbohydrate-active enzymes (CAZymes) when growing in a peptone-based medium supplemented with simple sugars and/or carboxymethyl cellulose (CMC) as carbon sources. This work aimed to identify the extracellular enzymatic cocktails through shotgun proteomics. The proteomic analysis showed that enzymes involved in cellulose and xylan degradation were among the most abundant proteins. These enzymes included an endo-glucanase GH5_2 and a glucuronoxylanase GH30_8, which were found in all conditions. In addition, several proteins were differentially expressed in the three evaluated culture media, indicating microbial metabolic changes due to the different supplied carbon sources, particularly, in the presence of CMC. Finally, the capability of the crude enzymatic cocktails from culture media to degrade birchwood xylan was assessed, which produced mostly xylooligosaccharides containing among 3–5 xylose units. Consequently, this work shows the potential of the extracellular enzymes from Bacillus sp. AR03 for producing emergent prebiotics.Fil: Hero, Johan Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Raimondo, Enzo Emanuel. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Martínez, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentin

Synergistic effect of xylanases produced in co-culture of bacillus sp. Ar03 and paenibacillus sp. Ar247

In nature, the plant biomass is degraded by a process that requires the cooperative action of multiple microorganisms capable of producing a variety of enzymes to attack the complex structure of lignocelluloses. This work assessed the production and the enzymatic activity over the main hemicellulolytic fraction of plant biomass, xylan, in monoculture and co-culture systems of bacteria isolated from regional niches associated with sugar cane bagasse. The enzyme activity was estimated by measuring reducing sugars released using the dinitrosalicylic acid method. All cultivation assays were performed at 200 rpm and 30 °C in a diluted peptone broth supplemented with 1% CMC (w/v). The viability and the growth of both isolates were estimated by the number of colony forming units, fact that was possible since both isolates exhibited different colony morphology. The specific xylanolytic activity of the co-culture of Bacillus sp. AR03 and Paenibacillus sp. AR247 was of 7.03 ± 0.46 IU/mg and 8.36 ± 0.49 IU/mg at 48 h and 96 h of cultivation, respectively. In contrast, each isolate assayed simultaneously under identical conditions, produced significantly lower xylanase activities, even when both isolates grew similarly in both, individual and co-cultures, reaching approximately 1011 CFU/ml in all cases. These values were of 4.18 ± 0.24 IU/mg and 4.55 ± 0.29 IU/mg of xylanolytic activity at 48 h and 96 h, respectively, for Bacillus sp. AR03, while Paenibacillus sp. AR247 reached values of 0.59 ± 0.09 IU/mg and 0.40 ± 0.03 IU/mg at the same periods of cultivation. When mixtures (1:1) of the cell-free supernatant of individual cultures were assayed, it was observed that the enzymatic activity reached a maximum of 4.16 ± 0.39 IU/mg after 48 h of cultivation. This value was close to that obtained by the sum of the enzymatic activity of individual cultures, which was 4.77 IU/mg, for the same cultivation time. The obtained results were consistent with the observation of a synergistic effect on the degradation of xylan in the co-culture evaluated, with an estimated degree of synergism of 1.69 at 96 h. This synergy, which has been described for enzyme mixtures on industrial substrates, was observed here during the co-cultivation of Bacillus sp. AR03 and Paenibacillus sp. AR247. This system displayed a higher xylanolytic activity with respect to the individual cultivation of each isolate and a different zymographic pattern along the cultivation period. The obtained results of the xylanolytic activity for individual strains and the co-culture might indicate that the observed effect could not depend on an only addition of enzyme activities so that we may suggest the existence of a synergistic cooperation during the growth in the co-cultivation of the microorganism evaluated.Fil: Hero, Johan Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Perotti, Nora Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Romero, Cintia Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaXI Congreso Argentino de Microbiología GeneralCórdobaArgentinaAsociación Civil de Microbiología Genera

Impact of biocontrol yeast clavispora lusitaniae 146 on the lemon microbiome

The use of biocontrol agents has been proposed as an effective alternative to reduce citrus decays for promoting sustainable agriculture based on organic fruit production. Among the different microbial biocontrol agents, Clavispora lusitaniae 146 stands out as it is able of effectively controlling green mold in lemons. Although there is growing recognition of the role that the microbiome plays in the health and physiology of many plant species, to date, the composition of the lemon microbiome is unknown, nor is the effect of yeast 146 on it. Thus, the aim of this research was to study the impacts of biocontrol yeast Clavispora lusitaneae 146 on the composition of the lemon microbiome. Lemons were harvested, and then divided into two treatments: untreated and treated lemons with biocontrol yeast C. lusitaneae 146. Fruits were then stored at room temperature for 7 days. DNA was extracted from a pool of 3 pieces of peel per sample, and used for PCR that amplified the bacterial hypervariable V3-V4 region of the 16S rRNA gene. Paired-end sequencing of amplicons was done on an Illumina MiSeq sequencer. To assess the effects of postharvest treatment and storage on the diversity of the lemon microbiome, we used a series of ANOVA and adonis (~PERMANOVA) models with Shannon diversity and community composition as the response variables, respectively. There was no statistically significant difference (KruskalWallies, p > 0.05) in bacterial diversity between the treated and untreated fruits. In this sense, the application of Clavispora lusitaneae 146 did not produce significant changes on bacterial communities of lemons during storage, including alpha diversity, community composition and structure. The bacterial community was dominated by roteobacteria,followed by Firmicutes and Actinobacteria. Specific bacterial taxa were only identified for untreated lemons: Methylobacteriaceae (Alphaproteobacteria) and unclassified bacteria, however in a low abundance. Here, we presented the first lemon microbiome and we showed that the microbial abundance, diversity, and community structures were not significantly different for both treatments, revealing that Clavispora lusitaniae 146 didn´t modify the native bacterial population of the fruit microbiome. The present study is part of larger project whose objectives are to define the complete lemon microbiome, assess the effects of the postharvest biocontrol agents on the composition of the lemon microbiome to develop a science-based strategy for manipulating this microbiome to prevent postharvest decay and physiological disorders.Fil: Rasuk, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Chacón, Florencia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pereyra, Martina María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Dib, Julian Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaXVIII Congreso de la Sociedad Argentina de Microbiología GeneralChapadmalalArgentinaSociedad Argentina de Microbiología Genera

Comparative oil extraction from mutt (Myliobatis goodei) liver by enzymatic hydrolysis: free versus immobilized biocatalyst

BACKGROUND: The development and fine-tuning of biotechnological processes for fish oil extraction constitute a very important focus to contribute to the development of a food industry based on fish consumption. This work lies in a comparative analysis of the oil extraction yield of Myliobatis goodei livers using free and immobilized enzymes. RESULTS: An immobilized biocatalyst was designed from the cell-free extract of a Bacillus sp. Mcn4. A complete factorial design was used to study the components of the bacterial culture medium and select the condition with the highest titers of extracellular enzymatic activities. Wheat bran had a significant effect on the culture medium composition for enzymatic production. The immobilized biocatalyst was designed by covalent binding of the proteins present in the cocktail retaining a percentage of different types of enzymatic activities (Mult.Enz@MgFe2O4). Among the biocatalyst used, Alcalase® 2.4 L and Purazyme® AS 60 L (free commercial proteases) showed extraction yields of 87.39% and 84.25%, respectively, while Mult.Enz@MgFe2O4 achieved a better one of 89.97%. The oils obtained did not show significant differences in their physical–chemical properties while regarding the fatty acid content, the oil extracted with Purazyme® AS 60 L showed a comparatively lower proportion of polyunsaturated fatty acids. CONCLUSIONS: Our results suggest that the use of by-products of M. goodei is a valid alternative and encourages the use of immobilized multienzyme biocatalysts for the treatment of complex substrates in the fishing industry.Fil: Morales, Andrés Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Gómez, María Inés. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Inorgánica; ArgentinaFil: Romero, Cintia Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Inorgánica; ArgentinaFil: Vittone, Marina. Instituto Nacional de Investigaciones y Desarrollo Pesquero; ArgentinaFil: Massa, Agueda Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina. Instituto Nacional de Investigaciones y Desarrollo Pesquero; ArgentinaFil: Lamas, Daniela Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina. Instituto Nacional de Investigaciones y Desarrollo Pesquero; Argentin

Novel xylan degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205

Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular β-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.Fil: Linares Pastén, Javier A. Lund University. Biotechnology Department; SueciaFil: Hero, Johan Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Teixeira, Cristina. Lund University. Biotechnology Department; SueciaFil: Nyman, Margareta. Department Food Technology, Engineering And Nutrition; SueciaFil: Adlercreutz, Patrick. Lund University. Biotechnology Department; SueciaFil: Martinez, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Nordberg Karlsson, Eva. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentin

Potencial biotecnológico de actinobacterias como fuente de enzimas de interés industrial

INTRODUCCIÓN: Las actinobacterias pertenecen a un grupo debacterias muy diverso que pueden vivir en ambientes terrestres y acuáticos,aunque predominan en suelos. Debido a que exhiben diversas propiedadesfisiológicas y metabólicas, la importancia de estas bacterias radica en su capacidadpara producir una amplia variedad de enzimas extracelulares, metabolitossecundarios (antibióticos, antivirales, antitumorales, fitotoxinas,biosurfactantes, inmunosupresores) y otros compuestos de interés industrial quesintetizan y liberan al medio. OBJETIVO: Evaluar la capacidad de actinobacterias procedentes dediferentes sitios prístinos y contaminados para producir enzimas extracelularesde interés industrial.MATERIALES YMÉTODOS: Se realizóun screening de actividades enzimáticas extracelulares, empleando unametodología de detección semi-cuantitativa en placas de Petri. Para ello, lascepas en estudio fueron sembradas por punción en diferentes medios de cultivoagarizados suplementados con sustratos como almidón, pectina, leche descremada,gelatina bacteriológica, Tween 80, Tween 20 y naringina, a fin de evaluar lacapacidad para producir enzimas con actividad amilasa, pectinasa, proteasa,gelatinasa, lipasa, esterasa y naringinasa, respectivamente. Las placas se incubarondurante 5 días a 30°C. Transcurrido este tiempo, las actividades enzimáticas seevidenciaron mediante la aparición de halos de hidrólisis/precipitaciónalrededor de las colonias bacterianas. El Índice Enzimático (IE) para cada cepase estimó a partir de la relación entre el diámetro del halo, observado comozona transparente o de opacidad (según el caso), y el diámetro de la colonia.RESULTADOS: De las 32 cepas evaluadas, se observó que todasprodujeron algún tipo de enzima extracelular, con la excepción de la cepa ER8que no expresó ninguna actividad enzimática en las condiciones y medios decultivo ensayados. La mayoría de las cepas presentó actividad gelatinasa(93,75%), mientras que la minoría presentó actividad pectinasa (68,75%). Además,los mayores valores de IE se detectaron para las actividades naringinasa ypectinasa. Es importante señalar que 19 cepas mostraron la capacidad deproducir las 7 actividades enzimáticas ensayadas, destacándose las cepas ER18,ER19 y ER20 por presentar valores de IE mayores a 2 en todos los casos, lo cualindicaría un buen nivel de producción de estas enzimas extracelulares en lascondiciones evaluadas.DISCUSIÓN Y CONCLUSIONES: Estos resultados demuestran el potencialbiotecnológico que tendrían estas actinobacterias para producir enzimashidrolíticas extracelulares con importantes aplicaciones en diversas industrias,particularmente en la industria alimentaria como aditivos en panaderías ycervecerías, en clarificación de jugos de frutas, en maduración de quesos, entreotros.Fil: Raimondo, Enzo Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Bazán, Lucas Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad San Pablo Tucumán; ArgentinaFil: Fuentes, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Benimeli, Claudia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina. Universidad Nacional de Catamarca. Facultad de Ciencias Exactas y Naturales; Argentina1° Congreso Nacional de Alimentos, Salud y AmbienteCórdobaArgentinaColegio de Licenciados y Técnicos en Química e Industrias de la Alimentación de la provincia de Córdob

Preliminary studies of the evolution of the sludge of an anaerobic reactor working with vinasse

Los reactores biológicos tipo UASB (reactores anaeróbicos de manto de lodo y flujo ascendente) son ampliamente usados en el tratamiento de efluentes líquidos de alta carga orgánica. Su diseño, apto para retener biomasa activa, permite mantener elevadas tasas de remoción de materia orgánica. Las principales características de los lodos presentes en estos biodigestores son la actividad metanogénica específica (AME), su concentración -expresada en términos de sólidos suspendidos volátiles (SSV)- y la estructura granular que adoptan en esta clase de biodigestores. Esta biomasa anaeróbica transforma la materia orgánica del efluente en biogás. En el presente trabajo se estudiaron los cambios producidos en la biomasa con respecto a los parámetros citados, en un reactor de 500 m3 de volumen que opera con vinaza. El monitoreo de los lodos se realizó antes, durante y después del período de funcionamiento del reactor (150 días). El primer muestreo se realizó 59 días antes del arranque, para determinar la actividad (AME) de los lodos que permanecieron en el reactor desde la zafra anterior. Este se consideró el AME inicial y fue de 0,20 gDQO/m3.d. Al cabo de 227 días, 18 después de haberse detenido el reactor, la actividad de los lodos fue de 0,61 gDQO/m3.d. La biomasa, medida en términos de SSV durante el funcionamiento del reactor (150 días), se incrementó desde 1,27 Kg/m3 a 6,41 Kg/m3, mientras que la granulometría de los lodos, en el mismo período, presentó una disminución de las fracciones de mayor tamaño.UASB (Upflow anaerobic sludge blanket) reactors are widely used in the treatment of wastewater with a high organic load. Its design, able to hold back active biomass, allows achieve high rate of removing organic matter. The main features of these sludge are specific methanogenic activity (SMA), their concentration, expressed as volatile suspended solids (VSS), and granular structure that this sludge take shape in these reactors. This anaerobic biomass is responsible for carrying out the transformation of organic matter into biogas. The present work was conducted to study the changes produced in biomass with respect to the parameters mentioned in a reactor of 500 m3 volume operating with vinasse. The sludge monitoring was performed before, during and after the period of reactor operation (150 days). First sampling was done 59 days before starting-up, to determine the activity´s sludge remaining in the reactor from the previous harvest. This one was considered the initial AME and was 0,20 gCOD / m3.d. After 227 days, 18 after the reactor have stopped, the activity of the sludge was 0,61 gCOD / m3.d. Biomass, measured in terms of volatile suspended solids (VSS), during reactor operation, increased from 1,27 kg/m3 to 6,41 kg/m3, while the particle size of the sludge showed an decrease of the fraction of high measurement.Fil: Molina, César Federico. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Montoya, Rebeca D. del Valle. No especifica;Fil: Pisa, José Horacio. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Machado, Walter D.. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; ArgentinaFil: Quaia, Eugenio A.. Gobierno de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial Obispo Colombres; Argentin

Biocatalysts from prokaryotes enzymes used in biorefinery applications. Genomic and technological studies.

La biomasa lignocelulósica contiene los polisacáridos renovables más abundantes de la naturaleza y su explotación no compite con los alimentos. Por lo tanto, es considerada como una materia prima adecuada para su utilización en industrias de base biológica, en las que las enzimas capaces de degradar y modificar carbohidratos son clave para su aprovechamiento en un contexto de biorrefinería. El eje principal de esta tesis se dirige a estudios fisiológicos y moleculares de bacterias productoras de enzimas carbohidrato-activas, así como a la producción y caracterización de estas últimas, como un aporte al desarrollo de biocatalizadores para tecnologías sustentables. A partir de una colección de procariotas autóctonos aislados de ambientes estratégicos, Cohnella sp. AR92 fue seleccionada por su novedosa filiación taxonómica y destacada actividad xilanolítica obtenida en un medio mineral suplementado con bagazo de caña de azúcar pretratado con álcali, un subproducto agroindustrial. Por medio de diseños factoriales, se optimizó la composición del medio de cultivo para la producción de xilanasas, alcanzándose 15,8 UI/mL, con un incremento del 20 % con respecto al medio inicial. Posteriormente, mediante el estudio de las condiciones operativas, la producción enzimática fue mejorada aproximadamente tres veces (45 UI/mL), cuando el medio óptimo se inoculó con 5*107 UFC/mL y se incubó a 200 rpm, pH 7,0 y 25 °C durante 96 h.El preparado xilanolítico obtenido fue estable a temperaturas moderadas (30 °C - 40 °C) dentro de un amplio rango de pH (4 a 10). Su actividad no fue modificada significativamente por una variedad de cationes y aditivos ensayados, y retuvo más del 50 % de actividad frente a NaCl 3 M y a etanol 10 % (v/v). El cóctel xilanolítico hidrolizó eficazmente las hemicelulosas presentes en los residuos lignocelulósicos de origen agroindustrial evaluados, rindiendo xilosa y xilooligómeros, lo cual evidencia su potencial utilidad en la el aprovechamiento integral de la biomasa vegetal.Cohnella sp. AR92 posee un genoma de 6,0 Mpb y un contenido de GC de 56,0 %. Al igual que otros miembros de Paenibacillaceae, contiene abundancia y redundancia de genes que codifican para enzimas carbohidrato-activas, algunos de los cuales se agrupan en clusters. Dentro de Cohnella spp., la cepa AR92 presenta una destacada diversidad de este tipo de enzimas. El secuenciado del proteoma intra y extracelular de la cepa AR92 frente a bagazo de caña de azúcar crudo, bagazo pretratado con álcali y salvado de trigo permitió comprobar que gran parte de las enzimas involucradas en la degradación del xilano fueron detectadas en los tres sustratos analizados, siendo mayoritarias en bagazo pretratado con álcali. La principal glicósido-hidrolasa encontrada fue un GH11, seguida de varias enzimas GH10 multimodulares. El clonado y caracterización de la endo-1,4-β-xilanasa GH11 reveló como productos mayoritarios de degradación del xilano los xilooligómeros X3 a X6, lo cual pone de manifiesto su potencial para la generación de moléculas prebióticas.The lignocellulosic biomass contains the most abundant renewable polysaccharides of nature whose exploitation does not compete with food. Consequently, it is considered as a suitable raw material for use in bio-based industries, where polysaccharide degrading and modifying enzymes are of key relevance within a biorefinery context. The main goal of this thesis is focus to physiological and molecular studies of carbohydrate-active enzyme bacterial producers as well as their enzyme assessment as a contribution towards the development of biocatalysts for sustainable technologies. Starting from a collection of autochthonous prokaryotes isolated from strategic environments, Cohnella sp. AR92 was selected for its novel taxonomic affiliation and outstanding xylanolytic activity obtained on a mineral medium supplemented with alkali pre-treated sugarcane bagasse, an agro-industrial by-product. The culture medium components that promoted the enzymatic production were optimized by means of statistical designs, leading to 15.8 IU/mL and an increment of 20 % with respect to the initial medium. Then, the operative conditions were analyzed, which allowed to achieve enzyme titers of 45 IU/mL on the optimal medium inoculated with 5*107 CFU/mL and incubated at 200 rpm, pH 7.0 and 25 °C for 96 h. The xylanolytic preparation obtained was stable at moderate temperatures (30 °C - 40 °C) within a wide range of pH (4 to 10). The enzyme activity was not significantly modified by a variety of cations and additives tested, and more than 50 % of the activity was also retained against 3M NaCl and 10 % ethanol (v/v). The xylanolytic cocktail effectively hydrolyzed the hemicelluloses of the lignocellulosic residues assayed, yielding xylose and xylooligomers, which constitutes an evidence of its potential for an integral utilization of the vegetal biomass. The genomic analysis of Cohnella sp. AR92 revealed a 6.0 Mpb genome size with a 56.0 % GC content. Its annotation showed abundance of genes encoding redundant carbohydrate-active enzymes, many of them were grouped into clusters, features also observed in other members of Paenibacillaceae. Within Cohnella spp., the strain AR92 presented a large diversity of this type of enzymes compared with related isolates. The intra and extracellular proteome of the strain was sequenced from cells developed on raw sugarcane bagasse, alkali pretreated sugarcane bagasse and wheat bran. In all the conditions assayed, most of the potential xylan degrading enzymes were detected, mainly on alkali pretreated sugarcane bagasse. The main glycoside hydrolase identified was GH11, followed by several multimodular GH10 enzymes. The further cloning and characterization of the endo-1,4-β-xylanase GH11 revealed xylooligomers from X3 to X6 as the major products of xylan degradation, results that disclosed its potential for the generation of prebiotic molecules.Fil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentin

Synergistic Effect of Simple Sugars and Carboxymethyl Cellulose on the Production of a Cellulolytic Cocktail from Bacillus sp. AR03 and Enzyme Activity Characterization

A cellulase-producing bacterium isolated from pulp and paper feedstock, Bacillus sp. AR03, was evaluated by means of a factorial design showing that peptone and carbohydrates were the main variables affecting enzyme production. Simple sugars, individually and combined with carboxymethyl cellulose (CMC), were further examined for their influence on cellulase production by strain AR03. Most of the mono and disaccharides assayed presented a synergistic effect with CMC. As a result, a peptone-based broth supplemented with 10 g/L sucrose and 10 g/L CMC maximized enzyme production after 96 h of cultivation. This medium was used to produce endoglucanases in a 1-L stirred tank reactor in batch mode at 30 °C, which reduced the fermentation period to 48 h and reaching 3.12 ± 0.02 IU/mL of enzyme activity. Bacillus sp. AR03 endoglucanases showed an optimum temperature of 60 °C and a pH of 6.0 with a wide range of pH stability. Furthermore, presence of 10 mM Mn2+ and 5 mM Co2+ produced an increase of enzyme activity (246.7 and 183.7 %, respectively), and remarkable tolerance to NaCl, Tween 80, and EDTA was also observed. According to our results, the properties of the cellulolytic cocktail from Bacillus sp. AR03 offer promising features in view of potential biorefinery applications.Fil: Manfredi, Adriana Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; ArgentinaFil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; ArgentinaFil: Valdeón, Daniel Horacio. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perotti, Nora Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnologia; ArgentinaFil: Martinez, Maria Alejandra. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentin