Structural and Functional Characterization of Two <i>Pennisetum</i> sp. Biomass during Ultrasono-Assisted Alkali Pretreatment and Enzymatic Hydrolysis for Understanding the Mechanism of Targeted Delignification and Enhanced Saccharification

The recalcitrance offered by lignocellulose to get converted into simple sugars makes its conversion process complicated, hence pretreatment is required prior to enzymatic hydrolysis and fermentation. Ultrasonication-assisted alkali pretreatment (UA-NaOH) was found to be an effective pretreatment for delignification and enzymatic hydrolysis of denanath grass (DG) and hybrid napier grasses (HNG) in terms of maximum delignification and reducing sugar production. To determine the mechanism of pretreatment and enzymatic hydrolysis, the structural and functional characterization of native and pretreated grass biomass were investigated using SEM (scanning electron microscope), FT-IR (Fourier transformation infrared) spectroscopy, TGA (thermal gravimetric analysis), DSC (differential scanning) spectroscopy, and solid state ¹³C CP/MAS NMR (cross-polarization magic angle spinning nuclear magnetic resonance) spectroscopy. The surface erosions, distorted surface morphology and deconstruction of the cell wall components of the <i>Pennisetum</i> sp. were detected by SEM. The differences in the intra- and intermolecular hydrogen bonds that make the crystalline and amorphous regions in the cellulose were detected by FT-IR. While TGA studies revealed higher phenolic content in untreated grass biomass, DSC patterns indicated the formation of laevoglucose in DG pretreated samples. Interestingly, the NMR studies revealed the presence of maximum aliphatic lignin components with absence of the aromatic lignins in both DG and HNG samples. NMR results also showed the presence of maximum hexosans and xylans revealing that the presence of aliphatic lignin components could be a helpful way of retaining the monosaccharides

MOESM2 of Xylan epitope profiling: an enhanced approach to study organ development-dependent changes in xylan structure, biosynthesis, and deposition in plant cell walls

Additional file 2: Figure S2. Heat map showing gene expression from publicly available microarray data [29] of xylan genes in different stages of Col-0 stem development from immature (D1), intermediate (D2-D3), and mature (D4) stems. Levels of low (white) and high expression (red) are shown on a log2 scale for each xylan gene

Colorimetric intensity values from immuno-dot-assays of epitopes within cell wall polymers extracted from <i>G. hirsutum</i> and <i>G. barbadense</i> fiber at 10, 19, 24, and 30 DPA by hot acidic water.

<p>Antibody probes used were: (<b>A</b>) CCRC-M1 recognizing fucosylated XG epitopes and (<b>B</b>) CCRC-M58 recognizing non-fucosylated XG epitopes. Data points are the means (± SE) of 3 biological replications, and asterisks indicate that means from the two species at that DPA are significantly different as determined by T test (**p≤0.01).</p

Cotton Fiber Cell Walls of <em>Gossypium hirsutum</em> and <em>Gossypium barbadense</em> Have Differences Related to Loosely-Bound Xyloglucan

<div><p>Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition, <em>Gossypium hirsutum</em> (<em>Gh</em>) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in <em>Gh</em> and <em>Gossypium barbadense</em> (<em>Gb</em>), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the <em>Gb</em> fiber had a prolonged elongation period and developed higher quality compared to <em>Gh</em> fiber. The <em>Gb</em> fibers had a CFML, but it was not directly required for fiber elongation because <em>Gb</em> fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of <em>Gb</em> fiber contained lower levels of xyloglucan compared to <em>Gh</em> fiber. Xyloglucan endo-hydrolase activity was also higher in <em>Gb</em> fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.</p> </div

MOESM1 of Physical and chemical differences between one-stage and two-stage hydrothermal pretreated hardwood substrates for use in cellulosic ethanol production

Additional file 1: Biphenyl assay for uronic acids protocol and listing of monoclonal antibody names and their corresponding recognized glycan groups

Fiber developmental progression between 10 to 50 DPA for <i>G. hirsutum</i> (<i>Gh</i>) and <i>G. barbadense</i> (<i>Gb</i>) documented by fiber measurements and gene expression.

<p>(<b>A, B</b>) Fiber length (hand measured) and fiber weight/seed (A) and the ratio of fiber weight to length (B). The timing of visible CFML degradation, which began ∼21 DPA near the onset of secondary wall deposition, is marked by arrows; see the data for <i>Gb</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056315#pone-0056315-g002" target="_blank">Figure 2</a> and for <i>Gh</i> published previously (Singh et al., 2009). The data points (± SE) are the means of fiber measurements from 3 to 5 bolls of different plants. (<b>C</b>) Quantitative reverse transcription PCR data showing the developmental shift in gene expression prior to the onset of secondary wall cellulose synthesis at ∼22 DPA. For both <i>G. hirsutum</i> and <i>G. barbadense</i> fiber, the expression of three secondary wall-associated CESA genes began a sharp increase and the expression of an expansin isoforms began a sharp decline at ∼17 DPA. Each data point is the mean from 3 biological replicates. The fiber growth data for <i>Gh</i> up to 32 DPA (A) are republished from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056315#pone.0056315.s001" target="_blank">Figure S1</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056315#pone.0056315-Singh1" target="_blank">[3]</a> (<a href="http://www.plantphysiol.org" target="_blank">http://www.plantphysiol.org</a>, Copyright American Society of Plant Biologists).</p

Transmission electron micrographs of cross-sectioned <i>G. barbadense</i> fiber at 10 to 24 DPA.

<p>The ‘fb#’ labels indicate 2 or 3 individual fibers in each view. (<b>A, B, C</b>) At 10 DPA, 17 DPA, and 19 DPA, adjacent fibers are joined together by the CFML, the outermost layer of the primary wall. In many regions, a thin continuous wall exists between adjacent fibers (arrows in A, B, C). However, there are also periodic bulges between fibers that are filled with CFML material (asterisks in A, B, C). (<b>D</b>) At 24 DPA during secondary wall (sw) deposition, the fibers have separated due to CFML degradation, leaving empty space between them. The 2 µm scale bar in D applies to all micrographs.</p

Glycome Profiling of sequential extracts prepared from multiple fiber samples representing major stages of cotton fiber development in <i>G. hirsutum</i> and <i>G. barbadense</i>.

<p>A vertical color-coded strip shows the clades of cell wall glycan-binding antibodies (as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056315#pone.0056315-Pattathil2" target="_blank">[21]</a>). Each successive cell wall solvent is shown on the bottom, with a split panel above each label showing the results for <i>Gh</i> and <i>Gb</i> fiber at 14 to 35 DPA. Absorbance values >0.10 typically are perceived as non-black. The bar graphs show the amount of cell wall material removed by each solvent from each sample (mg extracted/g AIR). The dotted white box outlines binding of XG-directed antibodies to the oxalate extracts, where the most striking differences between the two species were observed.</p

Fiber differentiation processes on the DPA analyzed in these experiments.^a.

a<p>Bold types highlights a major difference between species, specifically the prolonged elongation period for <i>G. barbadense</i> fiber.</p

Xyloglucan endo-hydrolase (XEH) activity in developing 10 to 30 DPA fibers of <i>G. hirsutum</i> and <i>G. barbadense</i>.

<p>Data points are the means (± SE) of 4 biological replications. The data for <i>Gh</i> are republished from Table SVI of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056315#pone.0056315-Singh1" target="_blank">[3]</a> (<a href="http://www.plantphysiol.org" target="_blank">http://www.plantphysiol.org</a>, Copyright American Society of Plant Biologists).</p