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In vitro buffering capacity changes of seven commodities under controlled moisture and heating conditions.

Effects of adding 0 or .25 g of water/g of feed and heating for 0, 20, or 40 h at 100 degrees C on buffering capacity of beet pulp, almond hulls, alfalfa hay, oat hay, wheat bran, oat bran, and barley were determined in vitro. Feeds were examined for ash, N, NDF, ADF, acid detergent lignin, total buffering capacity (pH 4 to 9), and physiological buffering capacity (pH 5.5 to 7). Minerals were measured at 0 g of water/g of feed, 0 h of heat and at .25 g of water/g of feed, 40 h of heat; differences were negligible. Total buffering capacity was high for alfalfa, oat hay, and almond hulls; low for barley and oat bran; and intermediate for beet pulp and wheat bran. Physiological buffering capacity was consistently lower than total buffering capacity, but differences varied among and within feeds. Moisture had little effect on buffering capacity, NDF, ADF, or lignin. Time of heating had a significant effect on buffering capacity, NDF, ADF, and lignin but not on N or ash. Heating increased buffering capacity in most cases. Lignin was a poorer predictor of buffering capacity than total ash across feeds but a better predictor within feed across treatments. Large errors could occur when using total ash to predict buffering capacity for feeds subjected to heat.

Animal Feed↗

Investigation on the chemical structure of nonextractable residues of the fungicide cyprodinil in spring wheat using 13C-C1-phenyl-cyprodinil on 13C-depleted plants-an alternative approach to investigate nonextractable residues.

13C-labelled cyprodinil was applied on 13C-depleted wheat plants with 27-fold field application rate. A control experiment applying same amounts of 14C-cyprodinil showed that main portions of the residues were detected in the cellulose (15% NER), hemicellulose (28.3% NER), and lignin fraction (23.3% NER). 16.7% were detected in water soluble polymers, 6% in both, pectin and protein fraction, and 4% in the starch containing fraction. Free cyprodinil was detectable by TLC in all fractions except lignin. A direct characterization of the residues in vivo by CP-MAS was not successful. Cell wall fractions were further analysed by liquid state NMR to determine the structure of the mobilized highly polymer/polar residues: Within lignin, where most of the residues were located at field application rate, neither intact cyprodinil nor its metabolites could not be detected. The 13C-label introduced was probably incorporated in the polymer as natural lignin monomers and thus are not considered as bound residues according to IUPAC definition.

Carbon Isotopes↗

[Stimulation of the fecal excretion of polychlorinated biphenyls (KC-600) by diets containing rice bran fiber and cholestyramine].

Among the eight kinds of dietary fiber tested, rice bran fiber (RBF) mostly bound Kanechlor 600 (PCB) in vitro. The PCB bound to dietary fiber was in proportion to the lignin content in dietary fiber. We investigated whether RBF, lignin, cholestyramine and their combination stimulate the fecal excretion of PCB in rats. In the group fed a diet containing 10% (w/w) RBF, fecal excretion of PCB was stimulated. In the group fed a diet containing 10% (w/w) RBF and 5% (w/w) cholestyramine, more PCB was excreted in the feces. However, the group fed a diet containing 10% (w/w) RBF and 5% (w/w) lignin excreted the same amount of PCB as the group fed a diet containing 10% (w/w) RBF. The total PCB excreted into the feces for 21 days in the groups fed 10% (w/w) RBF, 10% (w/w) RBF and 5% (w/w) lignin, 5% (w/w) cholestyramine and 10% (w/w) RBF and 5% (w/w) cholestyramine diet was 3.4, 3.8, 2.3 and 5.7 times, respectively, that of the control. The stool transit time of rats fed 10% (w/w) RBF and 5% (w/w) cholestyramine diet was one hour faster than that of rats fed a 5% (w/w) cholestyramine diet. Therefore, we concluded that RBF has the greatest effect when administered in combination with cholestyramine on the fecal excretion of PCB.

Animals↗

Oxidation of polycyclic aromatic hydrocarbons and dibenzo[p]-dioxins by Phanerochaete chrysosporium ligninase.

The lignin peroxidase (ligninase) of Phanerochaete chrysosporium catalyzes the oxidation of a variety of lignin-related compounds. Here we report that this enzyme also catalyzes the oxidation of certain aromatic pollutants and compounds related to them, including polycyclic aromatic hydrocarbons with ionization potentials less than or equal to approximately 7.55 eV. This result demonstrates that the H2O2-oxidized states of lignin peroxidase are more oxidizing than the analogous states of classical peroxidases. Experiments with pyrene as the substrate showed that pyrene-1,6-dione and pyrene-1,8-dione are the major oxidation products (84% of total as determined by high performance liquid chromatography), and gas chromatography/mass spectrometry analysis of ligninase-catalyzed pyrene oxidations done in the presence of H2(18)O showed that the quinone oxygens come from water. We found that whole cultures of P. chrysosporium also transiently oxidize pyrene to these quinones. Experiments with dibenzo[p]dioxin and 2-chlorodibenzo[p]dioxin showed that they are also substrates for ligninase. The immediate product of dibenzo[p]dioxin oxidation is the dibenzo[p]dioxin cation radical, which was observed in enzymatic reactions by its electron spin resonance and visible absorption spectra. The cation radical mechanism of ligninase thus applies not only to lignin, but also to other environmentally significant aromatics.

Basidiomycota↗

Comparative study of beans from vine and bush type of vegetative growth. Effect of storage on cell wall components as factors in increasing cooking time.

Four samples each of black beans representing two types of vegetative growth were collected from farmers' fields in four locations in Guatemala. Soon after collection, samples were stored at 4 degrees and 38 degrees C at ambient relative humidity and subsamples were withdrawn at 0, 45, 90 and 135 days of storage for determination of water absorption, cooking time and analysis of neutral- and acid detergent fiber, cellulose, hemicellulose and lignin. The fiber fraction analysis were done on samples of 0, 45 and 90 days of storage. Water absorption for all 4 samples of the bush type was similar at both storage T, however the samples stored at 38 degrees C and at 135 days absorbed more water than when stored at 4 degrees C. The 4 vine types of beans showed different water absorption rates, with two showing patterns similar to those beans of the bush type and two which did absorbed water at a very slow rate. For both types of beans stored at 4 degrees C, cooking time decreased from 0 to 135 days of storage. On the other hand for all bean samples of the two types cooking time increased when stored at 38 degrees C. Analysis of variance showed highly significant effects due to plant type, days of storage, temperature and locality, and for some interactions. Analysis of variance of the fiber fractions showed high significant differences for days of storage for NDF, ADF, cellulose, hemicellulose, and lignin. Plant type gave significant differences for cellulose and hemicellulose. Highly significant differences for hemicellulose were found for the interactions of type x days, type x temperature, locality x type, and type x days x temperature. The rate of synthesis of the 5 fractions were calculated by simple regression analysis. For the bush type of beans some synthesis occurred at 4 degrees C, but it was enhanced when stored at 38 degrees C. For vine type of beans at 4 degrees C relative high rates of synthesis were observed, which were higher at 38 degrees C for NDF, hemicellulose and lignin. Cooking time and fiber fraction contents were subjected to regression analysis. The correlations at 38 degrees C were higher than at 4 degrees C for all fractions for both types of beans, but statistical significance was obtained only for NDF, ADF and cellulose for vine type of beans. These data show therefore that synthesis of cell wall structure fractions, and not only lignin formation, are responsible for the increase in cooking time observed upon storage at high temperature.

Analysis of Variance↗

Resistance of Populus davidiana × P. bolleana overexpressing cinnamoyl-CoA reductase gene to Lymantria dispar larvae.

Lignin is a crucial defense phytochemical against phytophagous insects. Cinnamoyl-CoA reductase (CCR) is a key enzyme in lignin biosynthesis. In this study, transgenic Populus davidiana × P. bolleana overexpressing the PdbCCR gene were generated via Agrobacterium-mediated transformation. Successful integration of PdbCCR into the poplar genome was confirmed by PCR amplification and quantitative reverse transcription PCR (qRT-PCR). The lignin content in the transgenic poplar leaves was significantly higher than that in the wild poplar, and after L. dispar larvae fed on the transgenic poplar, the CCR activity was clearly induced. The L. dispar larvae grew slowly after feeding on transgenic poplar and the laccase, cellulase and three detoxifying enzymes were induced compared with larvae after feeding on wild-type poplar. The bioassay further revealed that transgenic poplar plants overexpressing PdbCCR showed a high level of resistance to L. dispar larvae. These results confirmed that PdbCCR is a candidate gene for breeding insect resistant poplar.

Populus↗

Integrated multi-omics analyses provide new insights into genomic variation landscape and regulatory network candidate genes associated with walnut endocarp.

Persian walnut (Juglans regia) is an economically important nut oil tree; the fruit has a hard endocarp/shell to protect seeds, thus playing a key role in its evolution, and the shell thickness is an important trait for walnut breeding. However, the genomic landscape and the gene regulatory networks associated with walnut shell development remain to be systematically elucidated. Here, we report a high-quality genome assembly of the walnut cultivar 'Xiangling' and construct a graphic structure pan-genome of eight Juglans species to reveal the genetic variations at the genome level. We re-sequence 285 accessions to characterize the genomic variation landscape. Through genome-wide association studies (GWAS), we identified 19 loci associated with more than 268 loci that underwent selection during walnut domestication and improvement. Multi-omics analyses, including transcriptomics, metabolomics, DNA methylation, and spatial transcriptomics across eleven developmental stages, revealed several candidate genes related to secondary cell biosynthesis and lignin accumulation. This integrated multi-omics approach revealed several candidate genes associated with secondary cell biosynthesis and lignin accumulation, such as UGP, MYB308, MYB83, NAC043, NAC073, CCoAOMT1, CCoAOMT7, CHS2, CESA7, LAC7, COBL4, and IRX12. Overexpression of JrUGP and JrMYB308 in Arabidopsis thaliana confirmed their roles in lignin biosynthesis and cell wall thickening. Consequently, our comprehensive multi-omics findings offer novel insights into walnut genetic variation and network regulation of endocarp development and shell thickness, which enable further genome-informed breeding strategies for walnut cultivar improvement.

Juglans↗

Evidence for demethylation of syringyl moieties in archaeological wood using pyrolysis-gas chromatography/mass spectrometry.

Archaeological oak (Quercus sp.) wood samples, ranging from 16(th) C. AD to 6000 BP, were studied using flash pyrolysis-gas chromatography/mass spectrometry to obtain insight into angiosperm lignin degradation. The pyrolysates revealed evidence of a number of 3-methoxy-1,2-benzenediol derivatives, methoxycatechols, directly related to 2,6-dimethoxyphenol, syringyl, moieties which are characteristic building blocks of angiosperm lignin. Mass spectra and mass chromatograms of these compounds are reported. The finding of these characteristic pyrolysis products in well-preserved archaeological wood provides unequivocal evidence that demethylation of syringyl units occurs very early in wood degradation. It is highly likely that the absence of abundant 3-methoxy-1, 2-benzenediols in degrading plant materials containing angiosperm lignin relates to the lability of these newly formed moieties.

Archaeology↗

Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics.

Previous optimization strategies for the bioconversion of lignocellulosics by steam explosion technologies have focused on the effects of temperature, pH, and treatment time, but have not accounted for changes in severity brought about by properties inherent in the starting feedstock. Consequently, this study evaluated the effects of chip properties, feedstock size (40-mesh, 1.5 x 1.5 cm, 5 x 5 cm), and moisture content (12% and 30%) on the overall bioconversion process, and more specifically on the efficacy of removal of recalcitrant lignin from the lignocellulosic substrates following steam explosion. Increasing chip size resulted in an improvement in the solids recovery, with concurrent increases in the water soluble, hemicellulose-derived sugar recovery (7.5%). This increased recovery is a result of a decrease in the "relative severity" of the pretreatment as chip size increases. Additionally, the decreased relative severity minimized the condensation of the recalcitrant residual lignin and therefore increased the efficacy of peroxide fractionation, where a 60% improvement in lignin removal was possible with chips of larger initial size. Similarly, increased initial moisture content reduced the relative severity of the pretreatment, generating improved solids and hemicellulose-derived carbohydrate recovery. Both increased chip size and higher initial moisture content results in a substrate that performs better during peroxide delignification, and consequently enzymatic hydrolysis. Furthermore, a post steam-explosion refining step increased hemicellulose-derived sugar recovery and was most effectively delignified (to as low as 6.5%). The refined substrate could be enzymatically hydrolyzed to very high levels (98%) and relatively fast rates (1.23 g/L/h).

Biotransformation↗

Direct visualization of straw cell walls by AFM.

The structural relationship of cellulose, hemicellulose, and lignin in plant cell walls is still a mystery needing to be explored. By using atomic force microscopy (AFM) the surface of straw at different layers was directly observed, and the structural characteristics were analyzed by topographic analysis and FT-IR spectra. It was found that a compact layer of wax covered the outside of the straw, which protects the straw from insects and microorganisms. At the boundary of the primary and second wall there appears a network structure of cellulose and hemicellulose, with some lignin localised on the surface of the network. It is consistent with the model of a cell wall suggested by Vincent. Inside the second cell wall, there is a layer mainly composed of a cellulose crystalline region. High-resolution AFM observation reveals that the crystalline structure consists of both triclinic and monoclinic unit cells. An AFM phase image showing the structural relation between cellulose microfibrils, hemicellulose, and lignin in the straw cell wall.

Cell Wall↗

Identifying residues in natural organic matter through spectral prediction and pattern matching of 2D NMR datasets.

This paper describes procedures for the generation of 2D NMR databases containing spectra predicted from chemical structures. These databases allow flexible searching via chemical structure, substructure or similarity of structure as well as spectral features. In this paper we use the biopolymer lignin as an example. Lignin is an important and relatively recalcitrant structural biopolymer present in the majority of plant biomass. We demonstrate how an accurate 2D NMR database of approximately 600 2D spectra of lignin fragments can be easily constructed, in approximately 2 days, and then subsequently show how some of these fragments can be identified in soil extracts through the use of various search tools and pattern recognition techniques. We demonstrate that once identified in one sample, similar residues are easily determined in other soil extracts. In theory, such an approach can be used for the analysis of any organic mixtures.

Benzopyrans↗

Carbon isotope ratio analysis of organic moieties from fossil mummified wood: establishing optimum conditions for off-line pyrolysis extraction using gas chromatography/mass spectrometry.

Mummified fossil wood was studied using off-line pyrolysis-gas chromatography/mass spectrometry to reveal detailed insights into the pyrolysis conditions that are needed to obtain simultaneously sufficient amounts of both cellulose and lignin markers for stable carbon isotope analyses. The off-line pyrolysis was applied at a range of temperatures (200, 250 and 300 degrees C) and times (1 and 2 h) to determine the optimum temperature and time that yielded the highest quantity of true markers for lignin and cellulose. Increasing the time from 1 to 2 h had no effect whereas increasing the temperature led to large differences. The products released during the low-temperature pyrolysis were mostly related to thermally labile moieties. Only at 300 degrees C were sufficient amounts of products released that represent true cellulose and lignin building blocks and which could be studied using gas chromatography/combustion isotope ratio mass spectrometry.

Alcohols↗

Reduction of quinones and radicals by a plasma membrane redox system of Phanerochaete chrysosporium.

Quinones which are produced during the mineralization of lignin and xenobiotics by the white rot fungus Phanerochaete chrysosporium were reduced by a plasma membrane redox system of the fungus. Both intracellular enzymes and the plasma membrane redox system were able to reduce 1,4-benzoquinone. However, no quinone reductase activity was observed with the extracellular culture fluid. The intracellular reductase activity had a pH optimum between 6.0 and 7.0 and a Km of 150 microM. Reduction of 1,4-benzoquinone by the plasma membrane redox system had a pH optimum between 7.5 and 8.5 and exhibited saturation kinetics (Km = 11 microM, Vmax = 16 nmol/min/mg mycelia dry weight). Ferricyanide totally inhibited the quinone reduction until the ferricyanide was completely reduced by the membrane. Radicals (chlorpromazine and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)) that can be generated by the lignin peroxidases were also reduced by the plasma membrane redox system. Reduction of the ABTS cation radical also totally inhibited quinone reduction until the radical was completely reduced. Finally, quinone reduction rates were identical after the reduction of ferricyanide, ABTS cation radical, or quinone, suggesting that the plasma membrane redox system may actually protect the fungus from oxidative damage from free radicals generated by the lignin degrading system.

Basidiomycota↗

Effect of dietary fibre on gallstone formation in hamsters.

This study aimed at investigating the effect of different sources of dietary fibre on gallstone formation in hamsters. The substances studied were pectin, lignin and psyllium hydrocolloid. The two latter compounds protected hamsters against cholesterol gallstone formation. Lignin resulted in a decrease of the deoxycholic acid conentration and in a rise of the cholic/chenodeoxycholic acid ratio. These changes which are similar to those observed with cholestyramine suggest that lignin acts as a bile acid sequestrant. Psyllium hydrocolloid effected a similar shift of the cholic/chenodeoxycholic acid ratio but it also resulted in a rise of the deoxycholic concentration. This latter finding is not compatible with a bile acid sequestering role of this compound. The addition of alcohol to the drinking-water resulted in the formation of stones rich in pigment. Under these conditions the tendency to form such stones was not checked by either of the investigated substances.

Animals↗

Effect of dietary fiber on proteolytic pancreatic enzymes in vitro.

Chymotrypsin, trypsin, carboxypeptidase A and B, elastase and enterokinase activities were measured in buffer solutions and in human duodenal juice after incubation with wheat bran, cellulose, guar gum, pectin, psyllium and lignin. The different types of dietary fiber led to inhibition of enzymatic activity in most experiments, e.g., lignin could totally ablish the activity of isolated trypsin and chymotrypsin. Only in enterokinase was there no influence. Inhibition depended on incubation time; the effect was proportional to fiber concentration and inversely related to enzyme level. Treatment of fiber with hydrochloric acid (pH 1.5) and heat (95 degrees C) destroyed inhibitory activity in some experiments. The effect of lignin on one enzyme (trypsin) was reduced by the addition of another enzyme (chymotrypsin). It is concluded that dietary fiber could affect digestion by inhibiting proteolytic pancreatic enzymes.

Chymotrypsin↗

Effect of pretreatment on simultaneous saccharification and fermentation of hardwood into acetone/butanol.

The effectiveness of pretreatments on hardwood substrate was investigated in connection with its subsequent conversion by simultaneous saccharification and fermentation (SSF), using Clostridium acetobutylicum. The main objectives of the pretreatment were to achieve efficient separation of lignin from carbohydrates, and to obtain maximum sugar yield on enzymatic hydrolysis of pretreated wood. Two methods have given promising results: (1) supercritical CO2-SO2 treatment, and (2) monoethanolamine (MEA) treatment. The MEA pretreatment removed above 90% of hardwood lignin while retaining 83% of carbohydrates. With CO2-SO2 pretreatment, the degree of lignin separation was lower. Under the scheme of SSF, the pretreated hardwood was converted to acetone, butanol, and ethanol (ABE) via single stage processing by cellulase enzyme system and C. acetobutylicum cells. The product yield in the process was such that 15 g of ABE/100 g of dry aspen wood was produced. In the overall process of SSF, the enzymatic hydrolysis was found to be the rate-limiting step. The ability of C. acetobutylicum to metabolize various 6-carbon and 5-carbon sugars resulted in efficient utilization of all available sugars from hardwood.

Acetone↗

Micromechanics of plant tissues beyond the linear-elastic range.

We investigated the relation between cell wall structure and the resulting mechanical characteristics of different plant tissues. Special attention was paid to the mechanical behaviour beyond the linear-elastic range, the underlying micromechanical processes and the fracture characteristics. The previously proposed model of reorientation and slippage of the cellulose microfibrils in the cell wall [H.-CH. Spatz et al. (1999) J Exp Biol 202:3269-3272) was supported and is here refined, using measurements of the changes in microfibrillar angle during straining. Our model explains the widespread phenomenon of stress-strain curves with two linear portions of different slope and sheds light on the micromechanical processes involved in viscoelasticity and plastic yield. We also analysed the velocity dependence of viscoelasticity under the perspective of the Kelvin model, resolving the measured viscoelasticity into functions of a velocity-dependent and a velocity-independent friction. The influence of lignin on the above-mentioned mechanical properties was examined by chemical lignin extraction from tissues of Aristolochia macrophylla Lam. and by the use of transgenic plants of Arabidopsis thaliana (L.) Heynh. with reduced lignin content. Additionally, the influence of extraction of hemicelluloses on the mechanical properties was investigated as well as a cell wall mutant of Arabidopsis with an altered configuration of the cellulose microfibrils.

Arabidopsis↗

Metabolic diversion of the phenylpropanoid pathway causes cell wall and morphological changes in transgenic tobacco stems.

Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.

Cell Wall↗