Hemicellulolytic bacteria in the anterior intestine of the earthworm Eisenia fetida (Sav.).

Tropical agriculture produces large amounts of lignocellulosic residues that can potentially be used as a natural source of value-added products. The complexity of lignocellulose makes industrial-scale processing difficult. New processing techniques must be developed to improve the yield and avoid this valuable resource going to waste. Hemicelluloses comprise a variety of polysaccharides with different backbone compositions and decorations (such as methylations and acetylations), and form part of an intricate framework that confers structural stability to the plant cell wall. Organisms that are able to degrade these biopolymers include earthworms (Eisenia fetida), which can rapidly decompose a wide variety of lignocellulosic substrates. This ability probably derives from enzymes and symbiotic microorganisms in the earthworm gut. In this work, two substrates with similar C/N ratios but different hemicellulose content were selected. Palm fibre and coffee husk have relatively high (28%) and low (5%) hemicellulose contents, respectively. A vermicomposting mixture was prepared for the earthworms to feed on by mixing a hemicellulose substrate with organic market waste. Xylanase activity was determined in earthworm gut and used as a selection criterion for the isolation of hemicellulose-degrading bacteria. Xylanase activity was similar for both substrates, even though their physicochemical properties principally pH and electrical conductivity, as shown by the MANOVA analysis) were different for the total duration of the experiment (120 days). Xylanolytic strains isolated from earthworm gut were identified by sequence analysis of the 16S rRNA gene. Our results indicate that the four Actinobacteria, two Proteobacteria, and one Firmicutes isolated are active participants of the xylanolytic degradation by microbiota in the intestine of E. fetida. Most bacteria were more active at pH 7 and 28 °C, and those with higher activities are reported as being facultatively anaerobic, coinciding with the microenvironment reported for the earthworm gut. Each strain had a different degradative capacity.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues / Bacillus subtilis con actividades de endocelulasa y exocelulasa aislado en la fase termofílica del compostaje con residuos de café

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57 -#9702;C and pH 5.5 and the other, a temperature of 61 -#9702;C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24 h until 144 h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

El objetivo de este estudio fue aislar, seleccionary caracterizar bacterias con actividad celulolítica a partir de 2 diferentes pilas de compostaje de residuos de café, una con temperatura interna de 57°C y pH 5,5; la otra con temperatura interna de 61 °C y pH 9,3. Se utilizaron medios de cultivo con carboximetilcelulosa y celulosa cristalina como únicas fuentes de carbono. La actividad enzimàtica fue evaluada por formación de halos de hidrólisis, producción de azúcares reductores y zimogramas. De 20 cepas aisladas, 3 presentaron mayor actividad enzimàtica y fueron identificadas como Bacillus subtilis sobre la base de sus características morfológicas, fisiológicas y bioquímicas y del análisis de las secuencias de la región 16S del ADNr. Los extractos enzimáticos de las 3 cepas seleccionadas presentaron actividad de exocelulasa y de endocelulasa, con máximos de 0,254 y 0,519 U/ml, respectivamente; la actividad de estas enzimas se mantuvo incluso a pH ácido (4,8) o básico (9,3) y a temperaturas de hasta 60 °C. Las actividades enzimáticas halladas en este estudio se ubican dentro de las más altas reportadas para celulasas producidas por bacterias del género Bacillus. En los zimogramas se demostró actividad de endocelulasa desde las 24h hasta las 144h de incubación. Asimismo, se reporta por primera vez el efecto del pH sobre la actividad de endocelulasa observado por zimogramas. Los resultados de este estudio abren la posibilidad de hacer uso de estas enzimas en la obtención de sustratos fermentables para la producción de energía a partir de los residuos generados en grandes cantidades por la agroindustria del café.

Bacillus subtilis with endocellulase and exocellulase activities isolated in the thermophilic phase from composting with coffee residues.

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57°C and pH 5.5 and the other, a temperature of 61°C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24h until 144h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

High Throughput Screening: Developed Techniques for Cellulolytic and Xylanolytic Activities Assay.

High throughput screening (HTS) is a powerful tool in biotechnology. The search for new or improved enzymes with suitable biochemical properties for industrial processes, has resulted in high efforts and research activities to develop new methodologies for activity screening. In this context, important advances have been achieved for the screening of cellulases and xylanases activities from wild and recombinant microorganisms, and from sequence databases. These enzymes have a wide range of industrial applications, including food, animal feed, textile, pulp and paper industries and detergents. Cellulases and xylanases along with pectinases, represent 20% of the world enzyme market. Recently, cellulases and xylanases have been used on fermentable sugars recovered from lignocellulosic biomass for second-generation biorefineries, aimed to produce chemical and biofuel platforms. As a result, HTS methods for biomass or biomass-degrading enzymes are gaining importance. This article presents evidence of the studies carried out for HTS of cellulase and xylanase activities.