Employing perichromism for probing the properties of carboxymethyl cellulose films: an expedient, accurate method for the determination of the degree of substitution of the biopolymer derivative

The properties of films of carboxymethyl cellulose, CMC, of different degree of substitution, DS, have been examined by the use of perichromic indicators (probes). The film properties that have been determined are: empirical polarity, E-T(33); "acidity", alpha; "basicity", beta; and dipolarity/polarizability, pi*. This has been achieved by employing the following perichromic probes: 4-nitroaniline, 4-nitroanisole, 4-nitro-N,N-dimethylaniline, and 2,6-dichloro-4-(2,4,6-triphenyl-pyridinium-1-yl)phenolate, WB. The correlations between both E-T(33)- or pi* and DS were found to be linear; that between beta and DS is a second order polynomial; no obvious correlation was found between alpha and DS. The polarities of CMC films are in the range of those of butyl alcohols. As models for CMC, we have employed cellulose plus CMC of high DS; oxidized cellulose with degree of oxidation = 0.5; sodium glucuronate. The former model behaved akin to CMC, but the plots of the perichromic properties versus DS showed different slopes/intercepts. FTIR data and molecular dynamics simulations on the solvation of WB have shown that this difference can be traced to more efficient hydrogen bonding between the film of the model and the probe. This affects the intra-molecular charge-transfer energy of the latter, leading to different responses to the variation of DS. Based on the excellent linear correlation between E-T(33) and DS, for CMC from different origins, we suggest that perichromism is a simple, accurate, and expedient alternative for the determination of DS of the biopolymer derivative.

Expression of genes from the lignin synthesis pathway in guineagrass genotypes differing in cell-wall digestibility

Rapid decline in cell-wall digestibility hinders efficient use of warm-season grasses. The objective of this study was to identify genes whose expressions are related to the slope of decline in cell-wall digestibility. Eleven guineagrass genotypes were harvested at three ages and classified according to fibre digestibility. Extreme genotypes were separated into groups with either FAST or SLOW decline in fibre digestibility. Expression of transcripts from six genes from the lignin synthesis pathway was quantified by real-time PCR. Fast decline in fibre digestibility was associated with higher DM yield after 90 d of regrowth. Apart from lower fibre digestibility and higher lignin content for the FAST group, there were no other differences between the two groups for the chemical composition of stems and leaves. Maturity affected differently the expression of two of the six genes, cinnamate 4-hydroxylase and caffeoyl-CoA O-methyltransferase (C4H and CCoAOMT). Genotypes with fast decline in fibre digestibility had greater increase in the expression of C4H and CCoAOMT from 30 to 60 d of regrowth, than genotypes with slower decline. Expression of C4H and CCoAOMT appears to be related to the decline in cell-wall digestibility with advance in maturity of guineagrass.

Structural features of lignin obtained at different alkaline oxidation conditions from sugarcane bagasse

Lignin is a macromolecule frequently obtained as residue during technological processing of biomass. Modifications in chemical structure of lignin generate valuable products, some with particular and unique characteristics. One of the available methods for modification of industrial lignin is oxidation by hydrogen peroxide. In this work, we conducted systematic studies of the oxidation process that were carried out at various pHs and oxidizing agent concentrations. Biophysical, biochemical, structural properties of the oxidized lignin were analyzed by UV spectrophotometry, Fourier transform infrared spectroscopy, scanning electron microscopy and small angle X-ray scattering. Our results reveal that lignin oxidized with 9.1% H(2)O(2) (m/v) at pH 13.3 has the highest fragmentation, oxidation degree and stability. Although this processing condition might be considered quite severe, we have concluded that the stability of the obtained oxidized lignin was greatly increased. Therefore, the identified processing conditions of oxidation may be of practical interest for industrial applications. (C) 2011 Elsevier B.V. All rights reserved.

RELATIONSHIP AMONG EXTRACTIVES, LIGNIN AND HOLOCELLULOSE CONTENTS WITH PERFORMANCE INDEX OF SEVEN WOOD SPECIES USED FOR BOWS OF STRING INSTRUMENTS

In this paper we investigate the influence of extractives, lignin and holocellulose contents on performance index (PI) of seven woods used or tested for violin bows. Woods with higher values of this index (PI = root MOE/rho, where MOE is modulus of elasticity and rho is density) have a higher bending stiffness at a given mass, which can be related to bow wood quality. Extractive content was negatively correlated with PI in Caesalpinia echinata, Hanclroanthus sp. and Astronium lecointei. In C. echinata holocellulose was positively correlated with PI. These results need to be further explored with more samples and by testing additional wood properties. Although the chemical constituents could provide an indication of quality, it is not possible to establish appropriate woods for bows solely by examining their chemical constituents.

Shape resonance spectra of lignin subunits

We report integral cross sections for elastic electron scattering by the lignin subunits phenol, guaiacol, and p-coumaryl alcohol. Our calculations employed the Schwinger multichannel method with pseudopotentials and indicate three to four pi* shape resonances for each of these systems, suggesting that low-energy electrons could efficiently transfer energy into the lignin matrix. We also discuss dissociation mechanisms based on the calculated cross sections, available experimental data, virtual orbital analysis, and the knowledge on electron interactions with biomolecules. Our results point out a physical-chemical basis for electron-driven biomass delignification. The latter would be an essential step for efficient biofuel production from lignocellulosic materials.

Chemical composition and enzymatic digestibility of sugarcane clones selected for varied lignin content

Abstract Background The recalcitrance of lignocellulosic materials is a major limitation for their conversion into fermentable sugars. Lignin depletion in new cultivars or transgenic plants has been identified as a way to diminish this recalcitrance. In this study, we assessed the success of a sugarcane breeding program in selecting sugarcane plants with low lignin content, and report the chemical composition and agronomic characteristics of eleven experimental hybrids and two reference samples. The enzymatic digestion of untreated and chemically delignified samples was evaluated to advance the performance of the sugarcane residue (bagasse) in cellulosic-ethanol production processes. Results The ranges for the percentages of glucan, hemicellulose, lignin, and extractive (based on oven-dry biomass) of the experimental hybrids and reference samples were 38% to 43%, 25% to 32%, 17% to 24%, and 1.6% to 7.5%, respectively. The samples with the smallest amounts of lignin did not produce the largest amounts of total polysaccharides. Instead, a variable increase in the mass of a number of components, including extractives, seemed to compensate for the reduction in lignin content. Hydroxycinnamic acids accounted for a significant part of the aromatic compounds in the samples, with p-coumaric acid predominating, whereas ferulic acid was present only in low amounts. Hydroxycinnamic acids with ester linkage to the hemicelluloses varied from 2.3% to 3.6%. The percentage of total hydroxycinnamic acids (including the fraction linked to lignin through ether linkages) varied from 5.0% to 9.2%, and correlated to some extent with the lignin content. These clones released up to 31% of glucose after 72 hours of digestion with commercial cellulases, whereas chemically delignified samples led to cellulose conversion values of more than 80%. However, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment. Conclusion Some of the experimental sugarcane hybrids did have the combined characteristics of high biomass and high sucrose production with low lignin content. Conversion of glucan to glucose by commercial cellulases was increased in the samples with low lignin content. Chemical delignification further increased the cellulose conversion to values of more than 80%. Thus, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

Abstract Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.

Indigestible cellulose and lignin in determining feces production and apparent digestibility in horses

Four crossbred geldings were used in a randomized blocks experimental design. The objective was to study the use of the internal markers indigestible cellulose (iCEL) and indigestible lignin (iLIG), obtained in situ (cattle) or in vivo (equine) to predict nutrient apparent digestibility in horses. Treatments consisted of different methodologies to determine digestibility: direct method with total feces collection (TC), and indirect method using internal markers iCEL and iLIG obtained either by in situ incubation in bovine rumen or in vivo (IV) using the mobile nylon bag (MNB) technique in horses. Feces production was 2.80 kg in DM, and average recovery rate (p > 0.05) was 101%. Nutrient digestibility coefficient (p > 0.05) estimates were adequately predicted by iCEL and iLIG, obtained in situ or in vivo, with average values of 52.63, 54.17, 64.90, 43.73 and 98.28% for dry matter, organic matter, crude protein, neutral detergent fiber and starch, respectively. It can be concluded that iCEL and iLIG may be obtained in vivo by MNB in horses consuming a forage-concentrate diet, to predict nutrient digestibility coefficients.

Biochemical Conversions of Lignocellulosic Biomass for Sustainable Fuel-Ethanol Production in the Upper Midwest

Biofuels are an increasingly important component of worldwide energy supply. This research aims to understand the pathways and impacts of biofuels production, and to improve these processes to make them more efficient. In Chapter 2, a life cycle assessment (LCA) is presented for cellulosic ethanol production from five potential feedstocks of regional importance to the upper Midwest - hybrid poplar, hybrid willow, switchgrass, diverse prairie grasses, and logging residues - according to the requirements of Renewable Fuel Standard (RFS). Direct land use change emissions are included for the conversion of abandoned agricultural land to feedstock production, and computer models of the conversion process are used in order to determine the effect of varying biomass composition on overall life cycle impacts. All scenarios analyzed here result in greater than 60% reduction in greenhouse gas emissions relative to petroleum gasoline. Land use change effects were found to contribute significantly to the overall emissions for the first 20 years after plantation establishment. Chapter 3 is an investigation of the effects of biomass mixtures on overall sugar recovery from the combined processes of dilute acid pretreatment and enzymatic hydrolysis. Biomass mixtures studied were aspen, a hardwood species well suited to biochemical processing; balsam, a high-lignin softwood species, and switchgrass, an herbaceous energy crop with high ash content. A matrix of three different dilute acid pretreatment severities and three different enzyme loading levels was used to characterize interactions between pretreatment and enzymatic hydrolysis. Maximum glucose yield for any species was 70% oftheoretical for switchgrass, and maximum xylose yield was 99.7% of theoretical for aspen. Supplemental β-glucosidase increased glucose yield from enzymatic hydrolysis by an average of 15%, and total sugar recoveries for mixtures could be predicted to within 4% by linear interpolation of the pure species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.

Lignin, phenols, and some chemical elements in aerosols and bottom sediments from the Tropical North Atlantic

A study of composition of biomarkers (lignin and phenols) in aerosols and bottom sediments from the Tropical North Atlantic was carried out. It was shown that organic matter of aerosols was mostly composed of products of terrestrial plants (arboreal fibers, pollen, and spores). Biomarker composition in the aerosols and in the bottom sediments was practically similar, which proved delivery of terrigenous organic matter to the ocean via the atmosphere.

n-Alkanes and lignin in bottom sediments of the West European Basin (area of the battleship Bismark wreck site)

Three bottom sediment cores were collected from the top, slope, and foot of a small topographic high located near the West European continental rise within the Porcupine abyssal plain at the battleship Bismark wreck site. Using high-efficient gas chromatography technique we determined content and examined molecular composition of n-alkane fraction of hydrocarbons and phenol compounds of lignin. n-Alkane and phenol concentrations in bottom sediments of all three cores were low both in values per unit mass of sediments and in organic matter composition that is typical for pelagic deposits of the World Ocean. They vary from 0.07 to 2.01 µg/g of dry sediment and from 0.0001 to 0.01% of TOC; phenol ranges are from 1.43 to 11.1 µg/g and from 0.03 to 0.6%. Non-uniform supply of terrigenous matter to the bottom under conditions of changes in sedimentation environment in different geological epochs is the principal reason for significant variations in n-alkane and lignin concentrations with depth in the cores. Lignin and its derivatives make the main contribution to formation of organic matter composition of the region in study. With respect to n-alkane and lignin concentrations organic matter of deposits of the West European Basin is composed of remains of higher plants and of autochtonous organic matter of marine flora; they have mixed terrigenous-autochtonous (terrigenous-planktonogenic) origin.

Lignin and chemical elements in the surface layer of bottom sediments from the Kandalaksha Bay of the White Sea

Chemical composition of the upper layer of sediments (0-1 cm) in the Kolvits and Knazhaya inlets, and also in the deep-water part of the Kandalaksha Bay is considered. It is shown that silts are richer in Fe, TOC, and heavy metals, than sands. The highest concentration of these elements is found in sediments under mixing zones of riverine and sea waters. Correlations of P, Zn, Cd, and Cu with iron are high, and correlations of Pb and Cu with organic carbon are also high. Very high concentration of Pb in the Kandalaksha Bay indicate technogenic pollution of sediments. Lignin makes significant contribution to formation of organic matter in the sediments. Composition of lignin in bottom sediments of the Kandalaksha Bay is defined by composition of lignin in soils and aerosols. Vanillin and syringyl structures prevail in molecular composition of lignin in bottom sediments. Their sources are coniferous vegetations, soils, and mosses. Ratios of certain types of phenol compounds indicate pollution of the upper layer of sediments by technogenic lignin. Lead and copper correlate well with this technogenic lignin.