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The efficient transformation of lignocellulosic biomass into fermentable sugars is essential for building bioeconomies. Sugarcane is an important agricultural crop in a number of Latin American countries, including Brazil and Argentina. Herein culms from two different sugarcane (SC384 and SC724) and two energy cane varieties (EC3116 and EC3118) bred in Argentina were evaluated for sustainable production of second-generation biofuels and green chemicals. Changes in the biomass crystallinity, structure, and morphology introduced by pretreatments were investigated using X-ray diffraction (DRX), confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) techniques. Enzymatic hydrolysis yields of untreated and pretreated sugarcane and energy cane culms were determined and correlated with physical analyses and chemical composition characterizations. Overall, after combined acid and alkali pretreatment, enzymatic convertibility was highly efficient for all studied sugarcane and energy cane varieties, reaching over 97% of theoretical conversion yields. High crystallinity indices and crystallite sizes of pretreated culms and SEM results and CLSM were consistent with the removal of lignin, solubilization of hemicellulose, and amorphous parts of lignocellulose imprinted by the pretreatments. High potential of culms from sugarcane and energy cane varieties cultivated in Argentina for sustainable production of renewable lignocellulosic sugars and their transformation into green chemicals and fuels was demonstrated.

Biomass conversion processes have become increasingly important to mitigate fossil fuel consumption and to increase the contribution of renewable fuels into the world energy matrix. In this study, Eucalyptus urograndis wood chips were pretreated by autocatalytic steam explosion to produce cellulosic ethanol after enzymatic hydrolysis and fermentation. These experiments were organized in a central composite rotatable design using temperatures and reaction times ranging from 174 to 216 °C and from 4 to 11 min, respectively. Mass yields, cellulose degree of polymerization, and glucose yields after enzymatic hydrolysis showed a linear correlation with pretreatment severity. The best condition was set at 210 °C for 5 min due to its higher glucose yield after pretreatment and enzymatic hydrolysis. Pectins were almost completely solubilized after pretreatment while galactoglucomannans were more resistant to acid hydrolysis than arabinoglucuronoxylans. Alkaline delignification led to 90.3% lignin removal from steam-exploded materials, but its effect on enzymatic hydrolysis was almost negligible. For the best pretreatment condition, analyses by confocal laser scanning microscopy and solid-state nuclear magnetic resonance revealed important changes in fiber morphology and chemical composition, respectively. Enzymatic hydrolysis at 4 wt % total solids with Cellic CTec2 and Cellic CTec3 (Novozymes) led to 22.4 and 27.8 g L–¹ glucose equivalents in 96 h, respectively, whose fermentation with a commercial strain of Saccharomyces cerevisiae led to ethanol productivities greater than 3.5 g L–¹ h–¹ in 6 h.

Lytic polysaccharide monooxygenases (LPMOs) have been introduced into industrial cocktails used for biomass saccharification due to their capacity to boost enzymatic conversion of recalcitrant cellulose. The genome of the thermotolerant ascomycete Aspergillus fumigatus encodes 7 genes for LPMOs that belong to auxiliary activity family 9 (AA9). Here, we cloned, successfully expressed and performed biochemical evaluation of two CBM-less A. fumigatus LPMOs (AfAA9A and AfAA9B). A high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis demonstrated that AfAA9A and AfAA9B are able to oxide cellulose at C1 and C1/C4 positions, respectively. Synergic effects of LPMOs (separately and in combination) with cellulases were investigated. Supplementation of Celluclast 1.5 L with a low concentration of AfAA9B improved in 20 % the saccharification of sugarcane bagasse pretreated by steam explosion (SEB), while AfAA9A did not improvethe saccharification. Analysis of the hydrolyzed biomass by confocal laser scanning microscopy (CLSM) showed the LPMOs are promoting lignin oxidation in the lignocellulosic material. This study complements the available results concerning the utilization of LPMOs in the enzymatic saccharification of lignocellulosic biomass.

Background Using globally abundant crop residues as a carbon source for energy generation and renewable chemicals production stand out as a promising solution to reduce current dependency on fossil fuels. In nature, such as in compost habitats, microbial communities efficiently degrade the available plant biomass using a diverse set of synergistic enzymes. However, deconstruction of lignocellulose remains a challenge for industry due to recalcitrant nature of the substrate and the inefficiency of the enzyme systems available, making the economic production of lignocellulosic biofuels difficult. Metatranscriptomic studies of microbial communities can unveil the metabolic functions employed by lignocellulolytic consortia and identify novel biocatalysts that could improve industrial lignocellulose conversion. Results In this study, a microbial community from compost was grown in minimal medium with sugarcane bagasse sugarcane bagasse as the sole carbon source. Solid-state nuclear magnetic resonance was used to monitor lignocellulose degradation; analysis of metatranscriptomic data led to the selection and functional characterization of several target genes, revealing the first glycoside hydrolase from Carbohydrate Active Enzyme family 11 with exo-1,4-β-xylanase activity. The xylanase crystal structure was resolved at 1.76 Å revealing the structural basis of exo-xylanase activity. Supplementation of a commercial cellulolytic enzyme cocktail with the xylanase showed improvement in Avicel hydrolysis in the presence of inhibitory xylooligomers. Conclusions This study demonstrated that composting microbiomes continue to be an excellent source of biotechnologically important enzymes by unveiling the diversity of enzymes involved in in situ lignocellulose degradation. Electronic supplementary material The online version of this article (10.1186/s13068-017-0944-4) contains supplementary material, which is available to authorized users.

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic β-1,4- and β-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 β-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis).

Significant improvements in pretreatments and a deeper understanding of changes in the physical structure and chemical composition of pretreated lignocellulosic materials are required to guarantee the technical and economic sustainability of second-generation ethanol technologies. In present work, several biophysical techniques were applied to characterize sugarcane bagasse samples that have been subjected to autocatalytic, phosphoric acid and sulfuric acid catalyzed steam explosion under equivalent combined severity factors (CSF). Confocal laser scanning microscopy and fluorescence lifetime imaging studies demonstrated unequal changes in lignin distribution in the plant cell wall that could be nicely correlated with substrate susceptibility to enzymatic hydrolysis. Furthermore, solid state nuclear magnetic resonance corroborated these observations by showing the differences in biomass composition primarily associated with hemicellulose removal, whereas X-ray diffraction analysis revealed changes in crystallinity indexes and average crystallite sizes. Thus, the impact of pretreatment temperature on the physical structure and chemical composition of CSF-equivalent steam-exploded sugarcane bagasse was identified as the key factor for developing substrate accessibility. This calls for caution in using combined severity factor as a chief parameter for comparison of different pretreatment conditions.

The enzymatic mixture containing Novozyme Cellic CTec2 spiked with xylanase derived from Thermomyces lanuginosus (1000 U xylanase per g of substrate) resulted in enhanced saccharification of native, hydrothermally and autohydrolytically pretreated sugarcane bagasse when compared to the benchmark Cellic CTec2. Hydrothermally pretreated bagasse upon hydrolysis with the enzymatic mixture yielded considerably higher levels of sugars as compared to benchmark Cellic CTec2, resulting in saccharification efficiencies of 85.7, 77.8 and 78.9% at 10, 15 and 20% (weight per volume) substrate loadings, respectively. Autohydrolysed and native sugarcane bagasse samples had lower saccharification yields, although xylanase addition to the benchmark cocktail still was beneficial. Chemical compositional analysis and detailed morphological examination of the substrates after pretreatments as well as the enzymatic hydrolysis reactions employing confocal laser scanning microscopy (CLSM), field emission scanning electron microscopy (FESEM), X-Ray diffraction (XRD) and solid state nuclear magnetic resonance (ssNMR) provided fundamental insights into the structural changes during deconstruction and shed light on how such changes affected the biomass enzyme hydrolysis.

Economical sustainability of cellulosic ethanol technology still requires considerable improvements in efficacies of both pretreatment and enzymatic hydrolysis steps. In this work a number of physical techniques were applied to characterize sugarcane bagasse samples that underwent hydrothermal and/or organosolv pretreatments under variable conditions and to correlated the observed changes with the efficiency of enzymatic hydrolysis. Confocal and field emission scanning electron microscopy studies revealed morphological changes in lignin distribution in the plant cell wall. The hydrothermal pretreatment caused a disorder in the arrangement of the lignin, whereas organosolv pretreatment partially removed lignin from bagasse and fraction of it redeposited at the surfaces of cellulose fibers. The delignification process was also analyzed by both chemical composition analysis and nuclear magnetic resonance. Pretreatment conditions leading to a significant increase of the efficiency of enzymatic hydrolysis were identified. Our studies open avenues for further biophysical investigations of pretreated lignocellulosic biomass, which could lead to its improved enzymatic hydrolysis.

Pretreatment is an important technological step for the lignocellulose conversion into fuels and biochemicals. Steam explosion (STEX) pretreatment is a fast method for affecting of plant cell wall architecture by a sharp pressure change at high temperatures. Here, eight different STEX conditions were applied to sugarcane bagasse (SCB) samples using auto-catalyzed and sulfuric acid catalyzed processes under a range of combined severity factors (CSFs; from 0.37 to 1.34). Our results showed that STEX of auto-catalyzed and sulfuric acid-catalyzed SCB led to the removal of 70–80 % and 82–92 % of xylose, respectively. Enzymatic hydrolysis of auto-catalyzed and sulfuric acid catalyzed bagasse was performed of washed and un-washed pretreated material to access the impact of soluble inhibitors on overall cellulosic sugars recovery. Auto-catalyzed bagasse after washing showed significantly higher glucan conversion (96%) than washed bagasse after pretreatment (62%). On the other hand, sulfuric acid-catalyzed bagasse after washing had 94% glucan conversion as opposed to unwashed material which again had significantly smaller conversion levels (73%). Physical properties and chemical composition were characterized using high performance liquid chromatography, X-ray diffraction, 13C solid-state nuclear magnetic resonance and confocal laser scanning microscopy. The obtained results contribute to understanding of an influence of the morphological changes resulting from pretreatments on the efficiency of enzymatic hydrolysis. We show that the changes in physical structure and chemical composition of the pretreated biomass might not be determined solely by combined severity factor of the pretreatments, but by the whole complexity of chemical modifications that different conditions of pretreatments introduce in the plant biomass samples.

In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with D-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with D-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two-orders of magnitude decrease of the enzyme kcat without significant modification in its Km, whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable. This article is protected by copyright. All rights reserved.