Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction.

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. KEY POINTS: • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

Synergic activity of Cel8Pa ß-1,4 endoglucanase and Bg1Pa ß-glucosidase from Paenibacillus xylanivorans A59 in beta-glucan conversion.

In the efficient bioconversion of polysaccharides from lignocellulosic biomass, endoglucanases and ß-glucosidases are key enzymes for the deconstruction of ß-glucans. In this work, we focused on a GH8 endoglucanase (Cel8Pa) and a GH1 ß-glucosidase (Bg1Pa) from Paenibacillus xylanivorans A59. Cel8Pa was active on a broad range of substrates, such as ß-glucan from barley (24.5 IU/mg), lichenan (17.9 IU/mg), phosphoric acid swollen cellulose (PASC) (9.7 IU/mg), carboxi-methylcellulose (CMC) (7.3 IU/mg), chitosan (1.4 IU/mg) and xylan (0.4 IU/mg). Bg1Pa was active on cellobiose (C2) and cello-oligosaccharides up to C6, releasing glucose as the main product. When both enzymes were used jointly, there was a synergic effect in the conversion rate of polysaccharides to glucose. Cel8Pa and Bg1Pa presented important properties for simultaneous saccharification and fermentation (SSF) processes in second generation bioethanol production, such as tolerance to high concentration of glucose and ethanol.

Paenibacillus xylanivorans sp. nov., a xylan-degrading bacterium isolated from decaying forest soil.

A xylanolytic bacterial strain, named A59T, was isolated from a forest soil consortium in southern Argentina. Strain A59T is a Gram-stain-positive, facultative anaerobic, endospore-forming and rod-shaped bacterium. Its optimal growth conditions are 30 °C (range, 28-37 °C), pH 7 (range, pH 5-10) and it tolerates up to 7 % of NaCl (range, 2-7 %). Chemotaxonomic analysis revealed that strain A59Tpossesses meso-diaminopimelic acid in the cell wall. It contains menaquinone MK-7 as the predominant isoprenoid quinone and the major fatty acid is anteiso-C15 : 0 (35.1 %), with a moderate amount of C16 : 0 (6.9 %). According to 16S RNA gene sequence analysis, the isolate is phylogenetically placed in the same cluster as Paenibacillus taichungensis BCRC 17757T (99.7 % nucleotide sequence identity) and Paenibacillus pabuli NBRC 13638T (99.1 %) and is closely related to Paenibacillus tundrae A10bT (98.8 %). However, phylogenetic studies based on the housekeeping gyrB gene placed A59T in a separate branch from all other related type strains. Furthermore, the results of whole genome average nucleotide identity analysis (gANI) with related type strains was lower than 91.10 % and the digital DNA-DNA hybridization value was lower than 44.30 %, which are below the threshold values for separating two species. The DNA G+C content was estimated as 46.09 mol%, based on genome sequencing. On the basis of these results, A59T represents a new species of the genus Paenibacillus, and we propose the name Paenibacillusxylanivorans sp. nov. The type strain is A59T (=DSM 107920T=NCIMB 15123T).

A thermostable GH8 endoglucanase of Enterobacter sp. R1 is suitable for ß-glucan deconstruction.

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.

EcXyl43 ß-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 ß-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of ß-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented ß-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

Identification of the main mechanisms involved in the tolerance and bioremediation of Cr(VI) by Bacillus sp. SFC 500-1E.

Chromium pollution is a problem that affects different areas worldwide and, therefore, must be solved. Bioremediation is a promising alternative to treat environmental contamination, but finding bacterial strains able to tolerate and remove different contaminants is a major challenge, since most co-polluted sites contain mixtures of organic and inorganic substances. In the present work, Bacillus sp. SFC 500-1E, isolated from the bacterial consortium SFC 500-1 native to tannery sediments, showed tolerance to various concentrations of different phenolic compounds and heavy metals, such as Cr(VI). This strain was able to efficiently remove Cr(VI), even in the presence of phenol. The detection of the chrA gene suggested that Cr(VI) extrusion could be a mechanism that allowed this strain to tolerate the heavy metal. However, reduction through cytosolic NADH-dependent chromate reductases may be the main mechanism involved in the remediation. The information provided in this study about the mechanisms through which Bacillus sp. SFC 500-1E removes Cr(VI) should be taken into account for the future application of this strain as a possible candidate to remediate contaminated environments.

Improvement of simultaneous Cr(VI) and phenol removal by an immobilised bacterial consortium and characterisation of biodegradation products.

Microbial bioremediation emerged some decades ago as an eco-friendly technology to restore polluted sites. Traditionally, the search for microorganisms suitable for bioremediation has been based on the selection of isolated strains able to remove a specific type of pollutant. However, this strategy has now become obsolete, since co-pollution is a global reality. Thus, current studies attempt to find bacterial cultures capable of coping with a mixture of organic and inorganic compounds. In this sense, the bacterial consortium SFC 500-1 has demonstrated efficiency for Cr(VI) and phenol removal, both of which are found in many industrial wastewaters. In the present study, the ability of SFC 500-1 for simultaneous removal was improved through its entrapment in a Ca-alginate matrix. This strategy led to an increased removal of Cr(VI), which was partially reduced to Cr(III). Immobilised cells were able to tolerate and degrade phenol up to 1,500mg/l at high rates, forming catechol and cis,cis-muconate as oxidation intermediates. Successful removal potential through 5 cycles of reuse, as well as after long-term storage, was another important advantage of the immobilised consortium. These characteristics make SFC 500-1 an interesting system for potential application in the biotreatment of co-polluted effluents.

Biochemical and molecular mechanisms involved in simultaneous phenol and Cr(VI) removal by Acinetobacter guillouiae SFC 500-1A.

Bioremediation has emerged as an environmental friendly strategy to deal with environmental pollution. Since the majority of polluted sites contain complex mixtures of inorganic and organic pollutants, it is important to find bacterial strains that can cope with multiple contaminants. In this work, a bacterial strain isolated from tannery sediments was identified as Acinetobacter guillouiae SFC 500-1A. This strain was able to simultaneously remove high phenol and Cr(VI) concentrations, and the mechanisms involved in such process were evaluated. The phenol biodegradation was catalized by a phenol-induced catechol 1,2-dioxygenase through an ortho-cleavage pathway. Also, NADH-dependent chromate reductase activity was measured in the cytosolic fraction. The ability of this strain to reduce Cr(VI) to Cr(III) was corroborated by detection of Cr(III) in cellular biomass after the removal process. While phenol did not affect significantly the chromate reductase activity, Cr(VI) was a major disruptor of catechol dioxygenase activity. Nevertheless, this activity was high even in presence of high Cr(VI) concentrations. Our results suggest the potential application of A. guillouiae SFC 500-1A for wastewaters treatment, and the obtained data provide the insights into the removal mechanisms, dynamics, and possible limitations of the bioremediation.

Brassica napus hairy roots and rhizobacteria for phenolic compounds removal.

Phenolic compounds are contaminants frequently found in water and soils. In the last years, some technologies such as phytoremediation have emerged to remediate contaminated sites. Plants alone are unable to completely degrade some pollutants; therefore, their association with rhizospheric bacteria has been proposed to increase phytoremediation potential, an approach called rhizoremediation. In this work, the ability of two rhizobacteria, Burkholderia kururiensis KP 23 and Agrobacterium rhizogenes LBA 9402, to tolerate and degrade phenolic compounds was evaluated. Both microorganisms were capable of tolerating high concentrations of phenol, 2,4-dichlorophenol (2,4-DCP), guaiacol, or pentachlorophenol (PCP), and degrading different concentrations of phenol and 2,4-DCP. Association of these bacterial strains with B. napus hairy roots, as model plant system, showed that the presence of both rhizospheric microorganisms, along with B. napus hairy roots, enhanced phenol degradation compared to B. napus hairy roots alone. These findings are interesting for future applications of these strains in phenol rhizoremediation processes, with whole plants, providing an efficient, economic, and sustainable remediation technology.