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BackgroundGrasses are lignocellulosic materials useful to supply the billion-tons annual requirement for renewable resources that aim to produce transportation fuels and a variety of chemicals. However, the polysaccharides contained in grass cell walls are built in a recalcitrant composite. Deconstruction of these cell walls is still a challenge for the energy-efficient and economically viable transformation of lignocellulosic materials. The varied tissue-specific distribution of cell wall components adds complexity to the origins of cell wall recalcitrance in grasses. This complexity usually led to empirically developed pretreatment processes to overcome recalcitrance. A further complication is that efficient pretreatment procedures generally treat the less recalcitrant tissues more than necessary, which results in the generation of undesirable biomass degradation products.ResultsSix different sugarcane hybrids were used as model grasses to evaluate the tissue-specific distribution of hemicelluloses and the role of these components in cell wall recalcitrance. Acetylated glucuronoarabinoxylan (GAX) occurs in all tissues. Mixed-linkage glucan (MLG) was relevant in the innermost regions of the sugarcane internodes (up to 15.4 % w/w), especially in the low-lignin content hybrids. Immunofluorescence microscopy showed that xylans predominated in vascular bundles, whereas MLG occurred mostly in the parenchyma cell walls from the pith region of the hybrids with low-lignin content. Evaluation of the digestibility of sugarcane polysaccharides by commercial enzymes indicated that the cell wall recalcitrance varied considerably along the internode regions and in the sugarcane hybrids. Pith regions of the hybrids with high MLG and low-lignin contents reached up to 85 % cellulose conversion after 72 h of hydrolysis, without any pretreatment.ConclusionsThe collective characteristics of the internode regions were related to the varied recalcitrance found in the samples. Components such as lignin and GAX were critical for the increased recalcitrance, but low cellulose crystallinity index, high MLG contents, and highly substituted GAX contributed to the generation of a less recalcitrant material.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0513-2) contains supplementary material, which is available to authorized users.

BackgroundSugar cane internodes can be divided diagonally into four fractions, of which the two innermost ones are the least recalcitrant pith and the moderately accessible pith-rind interface. These fractions differ in enzymatic hydrolyzability due to structural differences. In general, cellulose hydrolysis in plants is hindered by its physical interaction with hemicellulose and lignin. Lignin is believed to be linked covalently to hemicellulose through hydroxycinnamic acids, forming a compact matrix around the polysaccharides. Acetyl xylan esterase and three feruloyl esterases were evaluated for their potential to fragment the lignocellulosic network in sugar cane and to indirectly increase the accessibility of cellulose.ResultsThe hydrolyzability of the pith and pith-rind interface fractions of a low-lignin-containing sugar cane clone (H58) was compared to that of a reference cultivar (RC). Acetyl xylan esterase enhanced the rate and overall yield of cellulose and xylan hydrolysis in all four substrates. Of the three feruloyl esterases tested, only TsFaeC was capable of releasing p-coumaric acid, while AnFaeA and NcFaeD released ferulic acid from both the pith and interface fractions. Ferulic acid release was higher from the less recalcitrant clone (H58)/fraction (pith), whereas more p-coumaric acid was released from the clone (RC)/fraction (interface) with a higher lignin content. In addition, a compositional analysis of the four fractions revealed that p-coumaroyl content correlated with lignin, while feruloyl content correlated with arabinose content, suggesting different esterification patterns of these two hydroxycinnamic acids. Despite the extensive release of phenolic acids, feruloyl esterases only moderately promoted enzyme access to cellulose or xylan.ConclusionsAcetyl xylan esterase TrAXE was more efficient in enhancing the overall saccharification of sugar cane, compared to the feruloyl esterases AnFaeA, TsFaeC, and NcFaeD. The hydroxycinnamic acid composition of sugar cane fractions and the hydrolysis data together suggest that feruloyl groups are more likely to decorate xylan, while p-coumaroyl groups are rather linked to lignin. The three different feruloyl esterases had distinct product profiles on non-pretreated sugar cane substrate, indicating that sugar cane pith could function as a possible natural substrate for feruloyl esterase activity measurements. Hydrolysis data suggest that TsFaeC was able to release p-coumaroyl groups esterifying lignin.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0153-3) contains supplementary material, which is available to authorized users.

In vitro propagation of neem (Azadirachta indica A. Juss.) may offer an efficient alternative to seed propagation of this species. For optimization of in vitro propagation, different basal salt formulations, growth regulators, and culture container sealants (polytetrafluoroethylene hydrophobic membranes [PTFE]) were evaluated. Nodal segments cultured on Murashige and Skoog (MS) medium showed the highest shoot formation per explant (1.67). Explants cultured in flasks containing MS medium with 0.5 mg L −1 benzyladenine, 0.5 mg L −1 kinetin, and 0.05 mg L −1 naphthaleneacetic acid, and sealed with two PTFE membranes, produced the highest number of shoots (4.04). In contrast, explants cultured in flasks without membranes showed leaf chlorosis and senescence. For plant recovery, regenerants were acclimatized in a substrate of coconut fiber and eucalyptus bark (1:1) and showed 80% survival. Our results indicated that the use of flasks with vents was beneficial for in vitro propagation of this important plant.

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BackgroundThe recent discovery that LPMOs can work under anaerobic conditions when supplied with low amounts H2O2 opens the possibility of using LPMOs as enzyme aids in biogas reactors to increase methane yields from lignocellulosic materials. We have explored this possibility by studying anaerobic digestion of various lignocellulosic materials: Avicel, milled spruce and birch wood, and a lignin-rich hydrolysis residue from steam-exploded birch. The digestions were added LPMOs and various cellulolytic enzyme cocktails and were carried out with or without addition of H2O2.ResultsIn several cases, enzyme addition had a beneficial effect on methane production, which was partly due to components present in the enzyme preparations. It was possible to detect LPMO activity during the initial phases of the anaerobic digestions of Avicel, and in some cases LPMO activity could be correlated with improved methane production from lignocellulosic materials. However, a positive effect on methane production was only seen when LPMOs were added together with cellulases, and never upon addition of LPMOs only. Generally, the experimental outcomes showed substrate-dependent variations in process efficiency and the importance of LPMOs and added H2O2. These differences could relate to variations in the type and content of lignin, which again will affect the activity of the LPMO, the fate of the added H2O2 and the generation of potentially damaging reactive-oxygen species. The observed effects showed that the interplay between cellulases and LPMOs is important for the overall efficiency of the process.ConclusionThis study shows that it may be possible to harness the power of LPMOs in anaerobic digestion processes and improve biogas production, but also highlight the complexity of the reaction systems at hand. One complicating factor was that the enzymes themselves and other organic components in the enzyme preparations acted as substrates for biogas production, meaning that good control reactions were essential to detect effects caused by enzyme activity. As also observed during regular aerobic enzymatic digestion of lignocellulosic biomass, the type and contents of lignin in the substrates likely plays a major role in determining the impact of LPMOs and of cellulolytic enzymes in general. More work is needed to unravel the interplay between LPMOs, O2, H2O2, and the multitude of redox-active components found in anaerobic bioreactors degrading lignocellulosic substrates.

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