Lignin modification improves the biofuel production potential in transgenic Populus tomentosa

Lignin has been recognized for its negative impact on forage digestibility, tree pulping properties, and cellulosic biofuel production, although it is the major structural component of the secondarily thickened cell walls of vascular plants. Earlier studies have demonstrated that lignin modification improves forage digestibility and poplar pulping properties. To determine whether lignin modification has beneficial effect on saccharification of lignocellulosic biomass, we pretreated and then enzymatically hydrolyzed the mature wood from transgenic poplar plants that expressed the antisense transgenes of monolignol biosynthesis genes 4-coumarate: CoA ligase (4CL) or caffeoyl CoA 3-O-methyltransferase (CCoAOMT). Firstly, a long-term field trial was set up for the transgenic plants. Over five years, the reduced trend of lignin content remained stable in all transgenic lines. And a total lignin reduction of up to 10% did not alter the growth rate or biomass yield of the transgenic poplars. In the mature wood, suppression of CCoAOMT increased saccharification potential, but 4CL down-regulation had no significantly positive effect on saccharification. Sugar yield were negatively correlated with soluble lignin content of dried, extractive-free stem biomass. These results imply that lignin modification can facilitate the process of saccharification for biofuel production in tree crops.     

Relationship of physicochemical characteristics and hydrolyzed hydroxycinnamic acid profile of barley varieties and nutrient availability in ruminants

The objectives of this study were to (1) investigate the relationship between physicochemical characteristics (mean/median particle sizes, physical hull content) and hydrolyzed hydroxycinnamic acid profile (ferulic acid (FA), para-coumaric acid (pCA), and their ratio) of barley varieties and in situ rumen degradability in dairy cattle; and (2) investigate rumen degradation kinetics of FA and pCA of CDC barley varieties grown in western Canada. Barley variety had a significant effect (P < 0.05) on rumen undegradable fraction of DM, FA, pCA, neutral (NDF) and acid detergent fiber (ADF) at 12 and/or 24 h of rumen incubation. FA in barley grain was more degradable than the pCA (P < 0.05). There were no differences (P > 0.05) in effective degradability of DM (EDDM) and EDFA, but significant differences in EDpCA (P < 0.05). Barley hull was strongly correlated to NDF, ADF, ADL, hemicellulose and cellulose (R > 0.78, P < 0.001) and correlated to FA (R = 0.57, P < 0.05) but not to pCA (R = 0.42, P > 0.05) in original samples. FA and pCA were highly and positively correlated to NDF, ADF, ADL, hemicellulose, cellulose, mean/median particle sizes, and rumen indigestible DM, NDF and ADF at either 12 or 24 h (P < 0.05). Mean/median particle size of barley grain positively influenced the rumen indigestible DM, but not others (FA, pCA). The results implied that reduction of barley hull, FA and pCA contents could increase the degradability of barley grain in rumen. Multi-regression with best model variable selection analysis revealed that FA was the factor most inhibiting to DM degradability of barley in rumen, and was the most effective factor to predict DM degradability, while hull was the most effective factor to determine NDF degradability in rumen. Both hull and FA affected ADF degradability in the rumen. The results indicate that breeding or identifying barley varieties with lower hull and FA contents would result in higher degradability, higher energy density and higher quality barley and improve nutrient availability of barley.     

Colonization of rotation crops and weeds by the potato black dot pathogenColletotrichum coccodes

The rotation crops wheat, barley, oat, maize, soybean, rye, yellow mustard, alfalfa, and spring canola and weeds eastern black nightshade, velvetleaf, timothy grass, orchard grass, and Giant foxtail common to potato-growing areas in North America were used to study the host range ofColletotrichum coccodes, the causal agent of potato black dot. The fungus was isolated from nine of 14 rotation crops and weeds that were inoculated: yellow mustard, soybean, spring canola, alfalfa, oat, eastern black nightshade, velvetleaf, giant foxtail, and timothy grass. In all, colonization was highest in black nightshades (87%) and velvetleaf (80%). Among the rotation crops, colonization was highest on yellow mustard (59%) followed by spring canola (33%) and soybean (30%).Colletotrichum coccodes was not isolated from wheat, barley, rye, maize, or orchard grass. The results indicated that crops used for rotation with potato should be selected carefully to prevent the increase ofC. coccodes inoculum in the soil and that weeds may help maintain viable inoculum ofC. coccodes in the absence of potato. Based on these results we recommend that wheat, barley, maize, or rye be used in rotation with potato in areas whereC. coccodes is present in high levels in the soil.     

Sequential extractions and structural characterization of lignin with ethanol and alkali from bamboo (Neosinocalamus affinis)

A sequential process with the combination of ethanol and alkali aqueous solutions was utilized to extract lignin from bamboo (Neosinocalamus affinis), a potential lignocellulosic material. In this case, the successive treatments of dewaxed bamboo with 70% ethanol at 80 °C, 0.2 and 0.5 M NaOH, 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0, and 3.0 M NaOH at 50 °C, resulted in a total yield of acid-insoluble lignin fractions of 10.06%, corresponding to release of 62.25% original lignin from the cell walls. The lignin fractions obtained were then characterized by GPC, FT-IR, NMR spectroscopy, and sugar analysis. As compared to the alkali lignin fractions, the ethanol-soluble lignin fraction had a relatively higher molecular weight (2670 g/mol) and the content of carbohydrates primarily consisted of glucose 2.01% and xylose 1.90%. This suggested that the carbohydrate chains linked to lignin may increase the hydrodynamic volume of lignin and therefore increase the apparent molecular weight of the ethanol-soluble lignin. HSQC spectra analysis revealed that the alkali lignin fractions consisted mainly of β-O-4′ linkages combined with small amounts of β-β′, β-5′, β-1′ linkages, and p-hydroxycinnamyl alcohol end groups. Furthermore, minor amounts of esterified p-coumaric and ferulic acids were also detected in the lignins isolated.     

Genetic and Agronomic Variation in Arabinoxylan and Ferulic Acid Contents of Durum Wheat (Triticum durumL.) Grain and Its Milling Fractions

Total arabinoxylan (AXt), water-extractable arabinoxylan (WeAX) and ferulic acid (FA) from five cultivars of durum wheat (Triticum durumL.), grown under four different agronomic conditions, were measured. Among the varieties analysed, AXt, WeAX and FA contents ranged between 4·07%–6·02%, 0·37%–0·56% and 0·784 mg/g–7·98 mg/g, respectively. High genetic and agronomic variability was detected for AXt, WeAX and FA. AXt and FA increased sharply in milling products for extraction rates above 60%. FA was quantified in durum wheat milling fractions. High concentrations of FA esterified to cell-wall arabinoxylans were found in the aleurone layer (69% of total FA), germ and seedcoat (26·6% of total FA). Only trace amounts were detected in the starchy endosperm (1·4% of total FA). A highly significant correlation appeared between AXt and FA contents.     

Phytate Reduction in Whole Grains of Wheat,Rye, Barley and Oats after Hydrothermal Treatment

Whole grains of different cereals have traditionally been prepared with water and heat prior to dehulling, but knowledge of the effect on nutritional properties is limited. The aim of the present study was to investigate if phytate reduction occurred during hydrothermal treatment of whole grains. Wheat, rye, hulled and dehulled barley, hulled oats and naked oats were incubated with either water or acetate buffer (pH 4·8) at 55 °C for 24 h with the exception of oats, which were incubated at 37 °C. Phytate in wheat, rye and barley was reduced by 46–77% when water was used and by 84–99% when acetate buffer was used. The phytate reduction in oats was considerably less, 8–26%, but, after grinding and soaking, phytate was reduced by 72–77% in dehulled oats and by 88–94% in naked oats. Citric acid and citrate buffer was used for pH adjustment in some experiments, and their use resulted in less phytate reduction than when acetate and lactic acid were used. Wet-steeping of naked oats and naked barley in water at 53–57 °C for 20–30 min reduced the bacterial counts by 99·97%, and the addition of acid prevented bacterial growth during the incubations. It was concluded that cereals with reduced phytate content and good hygienic quality can be developed and produced using hydrothermal treatment of whole grains.     

Trypsin inhibitors and nutritive value in cereals

Chemical assays demonstrated that rye and barley cultivars contained relatively high levels of trypsin inhibitor activity as compared to oat and wheat cultivars, and there was a low degree of stability to prolonged wet heat treatment. In feeding trials with broiler chicks, incorporation of 67% raw barley or 50% raw rye in the rations enhanced feed intake and weight gains, and the marginal increases in pancreas weight were not reversed by feeding autoclaved cereals. Raw rye cultivars fed at the 75% level in mouse diets reduced weight gains, feed efficiency, protein digestibility, protein efficiency ratio and biological value. Autoclaving to inactivate trypsin inhibitors, or ether extraction to remove the resorcinols, failed to improve the nutritive value of rye diets for mice. It appeared that the protease inhibitors in the four cereals were relatively weak inhibitors of trypsin in the digestive system despite stability to dry heat and acid pH.     

Release of Ferulic Acid from Cereal Residues by Barley Enzymatic Extracts

The use of esterase- and xylanase-rich extracts of barley for the release of ferulic acid (FA) from barley spent grain and wheat bran is proposed here. Esterase activity was determined in 11 barley varieties and enriched extracts were prepared from one of the varieties with higher activity, Halcyon (7·3 mU/g barley). The esterase(s) extract alone was able to release low amounts of FA from spent grain and wheat bran. However, the presence of the xylanase(s)-containing extract increased the release of FA and demonstrated synergistic reactions between these enzymes. Maximum FA released by the combination of esterase(s) and xylanase(s) extracts was 26% and 13·3% from spent grain and wheat bran respectively after 36 h incubation. FA from spent grain and wheat bran was also released by barley esterase(s) extract and Trichoderma viride xylanase. Reducing sugar release followed a pattern similar to that for the release of FA. However, barley xylanase(s) appear more specific than T. viride xylanase for spent grain. These results demonstrate the potential use of economical barley enzymes for the release of ferulic acid from cell wall materials.     

Structural characterization and isolation of lignin and hemicelluloses from barley straw

Structural characterization and isolation of lignin and hemicelluloses from crops are very important for industrial utilization. In this paper, the sequential treatments of barley straw using 90% dioxane, 80% acidic dioxane, 100% dimethyl sulfoxide, and 8% NaOH released total 93% of original lignin and 87% of original hemicelluloses. The extractions with acidic dioxane and dimethyl sulfoxide produced the original hemicelluloses and high-condensed lignin mainly from the middle lamella. FT-IR and NMR analyses show that the hemicelluloses of barley straw contain acidic arabinxylans as the major polysaccharides, which are substituted by α-l-arabinofuranose, 4-O-methyl-glucuronic acid, acetyl group (DS = 0.13), and xylose at O-3 and/or O-2 of xylan, and lignin contains β-O-4′ as a predominant interunit linkage with high amounts of β-5′ and β-1′. The guaiacyl and syringyl units are more etherified, and the proportion of erythro-β-O-4′ is slightly higher than that of threo-β-O-4′ in the lignin of barley straw.     

Cell wall composition and degradability of stem tissue from lucerne divergently selected for lignin and in vitro dry-matter disappearance

Lucerne populations that had previously been divergently selected for acid detergent lignin (ADL) concentrations of whole herbage were additionally divergently selected for in vitro dry-matter disappearance (IVDMD) of basal stems. Parental plants of the four selected populations were intercrossed and the half-sib progeny evaluated in a replicated field trial in 1988 over two harvests. Cell wall composition of basal stems was determined for detergent fibre components and neutral sugars, uronic acids, Klason lignin and esterified and etherified phenolic acids of the total fibre fraction. Lignin polymer composition was measured by nitrobenzene oxidation. Degradability of cell wall polysaccharide components was determined by a 48-h in vitro ruminal fermentation. Cell wall composition was changed by both selection criteria. Klason lignin proportion of the total fibre did not differ as a result of selection, whereas the ADL concentration of neutral detergent fibre was different among selection groups. The cell wall polysaccharides of lucerne basal stems shifted towards more cellulose (glucose residues) and less hemicellulose (xylose residues) with selection for either low ADL or high IVDMD. While degradability of the cell wall polysaccharides was correlated with various measures of lignin composition and concentration, the results were variable and inconclusive. Surprisingly, Kiason lignin and ADL were positively correlated with cell wall polysaccharide degradability, and esterified ferulic acid was negatively correlated with degradation of the cell wall. Selection for herbage ADL and basal stem IVDMD of lucerne did alter cell wall degradation, but the associated changes in cell wall lignification were not consistently correlated with cell wall degradability.     

Comparative study of organosolv lignins from wheat straw

Dewaxed wheat straw was treated with acetic acid–H₂O (65/35, v/v), acetic acid–H₂O (80/20, v/v), acetic acid–H₂O (90/10, v/v), formic acid–acetic acid–H₂O (20/60/20, v/v/v), formic acid–acetic acid–H₂O (30/60/10, v/v/v), methanol–H₂O (60/40, v/v) and ethanol–H₂O (60/40, v/v) using 0.1% HCl as a catalyst at 85 °C for 4 h, in which 78.2, 80.0, 88.2, 89.4, 94.1, 23.5 and 37.4% of the original lignin, and 42.4, 58.7, 70.0, 65.1, 76.5, 14.2 and 22.2% of the original hemicelluloses was released, respectively. Lignins obtained were characterized by their content of hemicelluloses, composition of phenolic acids and aldehydes, molecular weight, thermal stability and by UV, Fourier transform infrared (FT-IR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The results showed that aqueous organic acid was more effective than aqueous organic alcohol for extensive delignification and selective fractionation of cellulose, lignin and hemicelluloses from the straw. In particular, the addition of formic acid gave a significant effect on the dissolution of lignin. All the acid-insoluble lignin fractions contained small amounts of contaminated hemicelluloses as shown by their content of neutral sugars, 0.9–4.3%, and had weight-average molecular weight between 3960 and 4340 g mol⁻¹. An increase in concentration of acetic acid or formic acid in organosolv resulted in an increment in release of guaiacyl units and in lignin condensation. However, the lignin preparations released during the treatment with aqueous organic alcohol without organic acid contained almost equal amounts of non-condensed guaiacyl and syringyl units with fewer p-hydroxyphenyl units. The β-O-4 ether bonds together with β-β, β-5 and 5-5′ carbon–carbon linkages were identified to be present in lignin substructures.     

Assessment of biochemical markers identified in wheat for monitoring barley grain tissue

The possible use of specific biochemical compounds identified in wheat grains was evaluated for monitoring barley grain tissues during fractionation. First barley grain anatomy was studied through microscopic observation and quantification of the relative proportion of each anatomical part in four distinct barley samples from both hulled and hulless genotypes. As expected from cereal phylogeny and irrespective of the possible presence of hull, common features were observed between barley and wheat grains, but the aleurone layer predominated in the outer layers. The specific location of the compounds identified in wheat was established. Phytic acid was specifically localized in the aleurone layer and alkylresorcinols in the composite layer containing the testa, even if their concentration differed from that observed in wheat grain tissues. Thus, these two markers identified in wheat can be used to monitor the corresponding barley tissues, independent of the presence of hulls. Conversely, phenolic compounds, either ferulic acid trimer or p-coumaric acid, cannot be used to monitor respectively the outer pericarp or the aleurone cell walls in barley grains. p-coumaric acid was identified as an efficient marker of the hull and could be used to distinguish hulled or hulless barley grains and to help monitor the dehulling process.     

Improved lignin properties and reactivity by modifications in the autohydrolysis process of aspen wood

Autohydrolysis of aspen wood breaks down lignin β-O-4 linkages via both acidolysis and homolytical cleavage. A simultaneous repolymerization by lignin–lignin condensation is the major reason for the low degree of delignification and the poor lignin reactivity. Modification of the autohydrolysis reaction by the addition of either NaOH or 2-naphthol changes the mechanism such that less acidolysis and fewer lignin–lignin condensation reactions take place, and an almost complete delignification can be achieved. The resulting isolated lignins have a low M_w, a light color and still contain some of the native β-O-4 linkages. Lignin from autohydrolysis in the presence of NaOH possesses more carboxylic acid groups, while the addition of 2-naphthol results in a lignin with a large amount of phenolic hydroxyl groups and incorporated naphthalene rings. These types of change are beneficial since the lignin structure becomes much more homogeneous with a greater potential for further upgrading and use.     

Thermochemical characterisation of straws and high yielding perennial grasses

The research is concerned with thermochemical characterisation of straws and high yielding perennial grasses. Crops selected for this study include wheat straw (Triticum aestivum), rape straw (Brassica napus), reed canary grass (Phalaris arundinacea) and switch grass (Panicum virgatum). Thermogravimetric analysis (TGA) was used to examine the distribution of char and volatiles during pyrolysis up to 900 °C. Utilising multi-heating rate thermogravimetric data, the Friedman iso-conversional kinetic method was used to determine pyrolysis kinetic parameters. Light and medium volatile decomposition products were investigated using pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) up to 520 °C. The 22 highest yielding identifiable cellulose, hemicellulose and lignin biomass markers were semi-quantified taking into consideration peak areas from GC chromatograms. Notable differences can be seen in butanedioic acid, dimethyl ester (hemicelluloses decomposition products), 2-methoxy-4-vinylphenol (lignin marker) and levoglucosan (intermediate pyrolytic decomposition product of cellulose) content when comparing perennial grasses with straw. From results presented in this study, perennial grasses such as switch grass, have the most attractive properties for fast pyrolysis processing. This is because of the observed high volatile yield content of 82.23%, heating value of 19.64 MJ/kg and the relatively low inorganic content.     

XIP-type endoxylanase inhibitors in different cereals

Highly pure XIP-type (for Xylanase Inhibiting Protein) endoxylanase inhibitor fractions were selectively obtained with a high yield from rye, durum wheat, barley, and maize extracts by affinity chromatography with immobilised Aspergillus niger endoxylanase Xyn1 following removal of the TAXI-type (for Triticum aestivum xylanase inhibitor) endoxylanase inhibitors by affinity chromatography with immobilised Bacillus subtilis endoxylanase XynA. No inhibitors belonging to the XIP family occur in rice, oats, and buckwheat. N-terminal amino acid sequences of the non-wheat XIP-type inhibitors were very similar or identical to those of wheat XIP-1, a chitinase homologue. The isolated inhibitors are basic, monomeric proteins of ca. 30 kDa with pI values of at least 8.5 (rye, durum wheat, and barley XIP) and ca. 7.0 (maize XIP). They are, in general, active against fungal endoxylanases and do not hydrolyse chitin. SDS–PAGE analysis and high-resolution cation exchange chromatography suggest the presence of multiple XIP-type isoinhibitors in the different cereals.     

秸秆还田对玉米生长发育及产量的影响

在沙质碳酸盐草甸土壤上,设置常量化肥、半量秸秆还田 常量化肥、全量秸秆还田 常量化肥三个处理,研究了秸秆还田对玉米生长发育及产量的影响。结果表明,与常量化肥相比,半量秸秆还田处理的植株抗倒伏能力增强,叶面积指数提高,叶绿素含量增加,并且显著提高了产量;而全量秸秆还田处理的各项指标与对照基本相近。     

Kinetics of organosolv delignification of fibre crop Arundo donax L

Kinetics of ethanol–alkali delignification of fibre crop Arundo donax L. (giant reed) has been studied. The improved approach for determination of the reaction rate constants by accurate quantification of lignin fractions with different reactivity during standard procedure of graphical differentiation was applied. Following to a simplified model, the delignification process was considered as a complex of n-parallel irreversible first-order reactions with similar final product and analysed as a multi-component reaction system. Three kinetically distinguishable lignin fractions of A. donax were revealed and quantified in proportion of approximately 61, 23 and 16% (as initial, bulk and residual lignin, respectively) and their effective degradation rate constants were determined for different pulping conditions. The proportion of lignin fractions was different from that reported for wood, but close to another crop—wheat straw, where the initial lignin fraction was also found as a major fraction (about 90%). The values of apparent activation energy were estimated respectively as 64, 89 and 96 kJ mol⁻¹, and were generally within the range of those reported for wood kraft and organosolv pulping. The simulation of ethanol–alkali delignification using found kinetic parameters showed the high reproducibility of experimental data on lignin removal, providing thereby the adequate test on validation of the suggested kinetic approach. The data reproducibility was substantially higher in comparison with conventional consecutive kinetic model (sum of square residuals (SQR) 0.0036 versus 0.0856).     

Co-composting of lignin to build up humic substances—Strategies in waste management to improve compost quality

Co-composting of different lignin sources with biogenic waste from the separate collection in Vienna was carried out in lab-scale experiments to promote humification as lignin is known to be a precursor molecule of humic substances. Humification is a target to be reached by composting due to the favorable properties for soils and plants. Extractable humic acids have been proved to be a suitable parameter to assess humification. Biogenic waste was used as a reference and basic material for co-composting of lignin. Lignin from different origins and composition was added: microbiologically degraded wood and lignin powder from industrial processes. Preliminary results show that addition of 2–5% of lignin powder could improve humic acid formation. Microbiologically degraded wood did not effect higher humic acid contents within the short time of lab-scale composting.Humic acid determination is based on photometrical investigations. To ensure that humic acid contents were not pretended by the presence of lignin, quality control was carried out using FT-IR spectroscopy and thermal analysis. The results obtained showed that lignin had been integrated in the humic acid molecule.     

Proteolysis and amino acid degradation during ensilage of untreated or formic acid-treated lucerne and maize

Lucerne ( Medicago sativa ), field wilted to 330 g dry matter (DM) kg⁻¹, and whole-plant maize ( Zea mays ), 383 g DM kg⁻¹, were treated with formic acid (FA) at the rate of 4-5 1 t⁻¹ fresh forage. The effects of FA treatment on proteolysis and amino acid degradation was investigated at several intervals over a 90-d period of ensilage in laboratory silos. Formic acid treatment produced different patterns of pH decline during ensilage of the two forages, suggesting some degree of crop specificity in response to FA treatment. After 7 d of ensilage of lucerne there were increases of 81% in nonprotein nitrogen (NPN), and 104% in free amino acid nitrogen (FAA-N), but with FA-treated lucerne the increases were 39% and 31%, respectively. FA treatment resulted in a reduction in the concentration of most free amino acids in lucerne silage, with the exception of glutamic acid and serine; the branched chain amino acids as well as glycine, tyrosine and proline were significantly (P<0·05) reduced after 3 d of ensilage. By contrast, FA treatment of maize did not significantly ( p <0·05) affect the NPN content nor was there a consistent pattern in the changes in FAA-N. Formic acid treatment of maize did produce a significant reduction ( P <0·05) in the concentration of most individual free amino acids at 90 d of ensilage. Overall, FA treatment had a more predictable effect on proteolysis and amino acid degradation in ensiled lucerne than in ensiled whole-plant maize.     

Effect of Boiling in Water of Barley and Buckwheat Groats on the Antioxidant Properties and Dietary Fiber Composition

In recent years, there has been an ever-increasing interest in the research of polyphenols obtained from dietary sources, and their antioxidative properties. The purpose of this study was to determine the effect of boiling buckwheat and barley groats on the antioxidant properties and dietary fiber composition. Antioxidative properties were investigated using methyl linoleate model system, by assessing the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity and metal chelating activity. The results were compared with butylated hydroxytoluene (BHT). Raw barley and buckwheat groats extracts showed higher DPPH scavenging ability compared to boiled barley and buckwheat groats extracts. Raw barley groats extract exhibited higher antioxidant activity than boiled groats extract in the methyl linoleate emulsion. Higher chelating ability in relation to Fe (II) ions was observed for boiled groats extracts as compared to raw groats extracts. BHT showed small antiradical activity and metal chelating activity, while showing higher antioxidative activity in emulsion system. The analysis of groats extracts using HPLC method showed the presence of rutin, catechin, quercetin, gallic, p-hydroxybenzoic, p-coumaric, o-coumaric, vanillic, sinapic, and ferulic acids. Differences in the content of dietary fiber and its fractions were observed in the examined products. The highest total dietary fiber content was detected in boiled buckwheat groats, while the lowest - in boiled barley groats. The scientific achievements of this research could help consumers to choose those cereal products available on the market, such as barley and buckwheat groats, which are a rich source of antioxidative compounds and dietary fiber.