Exploring the cellulolytic activity of environmental mycobacteria.

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.

Evaluation of different nitrogen sources on growth and fermentation performance for enhancing ethanol production by wine yeasts.

The utilization of grape juice from low oenological value grape varieties for bioethanol production represent an alternative for diversification and value addition in viticulture. Optimizing Very High Gravity (VHG) fermentation can significantly increase ethanol productivity while reducing water and energy consumption. In this study, the impact of different nitrogen sources on growth and fermentative performance of locally selected yeast strains was investigated. Five yeast strains of species Saccharomyces cerevisiae and Zygosaccharomyces rouxii were cultured in both synthetic culture media and natural grape juice supplemented with ammonium sulfate (NH), yeast extract (YE), Fermaid K (FERM), and urea (U) at varying concentrations. Due to the very low fermentation rate, the Z. rouxii strain was excluded from the selection. The results obtained in synthetic medium showed that nitrogen sources that promoted growth (NH and YE) had minimal effects on fermentative performance and were highly dependent on the specific yeast strain. However, the combination of urea and ammonium favored the rate of sugar consumption. When validated in natural grape juice, urea combined with ammonium (U + NH 300 + 75 mg/L) improved both growth parameters and ethanol yield. Doubling the concentration (U + NH 600 + 150 mg/L) further enhanced sugar consumption and ethanol production while reducing unwanted by-products. The combined use of urea and ammonium exhibited a synergistic effect, making it a cost-effective nitrogen supplement for VHG fermentations.

Characterization of a novel GH10 alkali-thermostable xylanase from a termite microbiome.

The aim of the present study was to assess the biochemical and molecular structural characteristics of a novel alkali-thermostable GH10 xylanase (Xyl10B) identified in a termite gut microbiome by a shotgun metagenomic approach. This endoxylanase candidate was amplified, cloned, heterologously expressed in Escherichia coli and purified. The recombinant enzyme was active at a broad range of temperatures (37-60 ºC) and pH values (4-10), with optimal activity at 50 ºC and pH 9. Moreover, its activity remained at more than 80% of its maximum at 50 °C for 8 h. In addition, Xyl10B was found to be stable in the presence of salt and several ions and chemical reagents frequently used in the industry. These characteristics make this enzyme an interesting candidate for pulp and paper bleaching industries, since this process requires enzymes without cellulase activity and resistant to high temperatures and alkaline pH (thermo-alkaliphilic enzymes). The products of xylan hydrolysis by Xyl10B (short xylooligosaccharides, xylose and xylobiose) could be suitable for application as prebiotics and in the production of bioethanol.

Evidencia molecular de la ocurrencia de bacterias celulolíticas del género Cohnella en especies neotropicales de Nasutitermitinae colectadas en el NEA / Molecular evidence that cellulolytic bacterial genus Cohnella is widespread among Neotropical Nasutitermitinae from NE Argentina

Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.

Cohnella es un género de bacterias celulolíticas que puede ser encontrado en una variedad de hábitats. El propósito de este estudio fue registrar su presencia en el tracto digestivo de termitas (Nasutitermes aquilinus, N. corniger y Cortaritermes fulviceps) colectadas en el noreste argentino (NEA). Se incubaron homogenados de intestinos en sustrato celulósico para multiplicar las bacterias. Utilizando nuevos cebadores para Cohnella spp., se amplificó una porción del ADN ribosomal 16S bacteriano, el cual fue posteriormente clonado. Las secuencias obtenidas mostraron su mayor porcentaje de similitud (97,2-99,9%) con Cohnella spp., previamente reportadas en diversos ambientes. El análisis filogenético tendió a agrupar a los clones de acuerdo a la especie hospedante y al sitio de muestreo. Estos resultados indican que especies de termitas frecuentes en el NEA albergan simbiontes intestinales relacionados con el género Cohnella. Las potenciales aplicaciones industriales de estos microorganismos animan a profundizar los estudios.

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and cycling-primed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect C-PRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of chromosomes containing key genes and/or QTL associated to agronomic important traits in sunflower.

Genetic mapping of EST-SSR, SSR and InDel to improve saturation of genomic regions in a previously developed sunflower map

In order to saturate a sunflower genetic map and facilitate marker-assisted selection (MAS) breeding for stress response, it is necessary to enhance map saturation with molecular markers localized in linkage groups associated to genomic regions involved in these traits. This work describes the identification and characterization of 1,134 simple sequence repeat (SSR) containing expressed sequence tags (EST) from unigenes available databases. Twelve of these functional markers as well as 41 public SSR markers were successfully localized in linkage groups, thus contributing to the saturation of specific regions on a reference genetic-linkage-map derived from recombinant inbred lines (RIL) mapping population from the cross between PAC2 x RHA266 lines. The enriched map includes 547 markers (231 SSR, 9 EST-SSR, 3 insertions/deletions (InDel) and 304 amplified fragment length polymorphisms (AFLP) distributed in 17 linkage groups (LG), spanning genetic size to 1,942.3 cM and improving its mean density to 3.6 cM per locus. As consequence, no gaps longer than 13.2 cM remain uncovered throughout the entire map, which increases the feasibility of detecting genes or traits of agronomic importance in sunflower.