Acetyl Bromide Lignin Research Articles

Spectrophotometric Determination of Biopolymers in Alabama Benchmark Soils

Biopolymers are organic polymers synthesized by biological organisms. Cellulose, lignin, and proteins are important soil biopolymers known to bind soil particles to improve or strengthen soil structures and support agricultural productivity. In this study, we spectrophotometrically determined the amount of soil cellulose, acetyl bromide lignin, and autoclaved citrate extractable protein in soils in relation to other soil properties. Results showed acetyl bromide lignin > cellulose > autoclaved citrate extractable protein. High clay soils have relatively higher amounts of cellulose and lignin but have lower protein content. The coefficient of variation (CV) of the three biopolymers in the soils studied was autoclaved citrate extractable protein (56.8%), >cellulose (55.2%), >acetyl bromide lignin (44.7%). Pearson correlation analysis showed that soil cellulose was significantly related to cation exchange capacity (CEC), total nitrogen, soil organic matter, and available phosphorus. An increase in soil acetyl bromide lignin suggested an increase in soil organic matter and lower soil available phosphorus. Soil autoclaved citrate extractable protein was significantly correlated with extractable acidity. However, cellulose, acetyl bromide lignin, and autoclaved citrate extractable protein were not significantly correlated with permanganate oxidizable carbon (POxC), electrical conductivity (EC), and C:N ratio. We assume that the concentrations of biopolymers in soils are an intrinsic soil characteristic and contribute to general soil health and productivity.

Assessing the Repeatability and Reliability of NIRS to Predict Nutritional Values and to Evaluate Two Lignin Methods in Urochloa spp. Grasses

Reliable forage analysis is crucial for proper ration formulation of ruminant herds. Through its fast, inexpensive, and non-destructive procedures, near-infrared spectroscopy (NIRS) has become a valuable method for forage evaluating. Notwithstanding, NIRS needs calibration before routine analysis. In addition, to evaluate the best method for lignin quantification in Urochloa spp. grasses is crucial under a digestibility perspective in grass-fed ruminant production. The aims of this study were to use 149 samples from different Urochloa species to develop NIRS calibration curves (partial least squares regressions) for acid detergent lignin (ADL), acetyl bromide lignin (ABL), as well as for ash, cell wall (CW), neutral detergent fiber (NDF), acid detergent fiber (ADF), in vitro DM digestibility (IVDMD), and in vitro NDF digestibility (IVNDFD). Moreover, the aim of this study was to correlate the in vitro digestibility with lignin quantification methods: ADL and ABL. Near-infrared spectroscopy showed potential for the quantification of Urochloa spp. properties, such as lignin contents (ADL and ABL) and ash, CW, NDF, ADF, IVDMD, and IVNDFD. However, calibrations performed using NIRS to measure ADF, ADL, IVDMD, and IVNDFD need to be thought about with caution before their utilization as a routine analysis for determining the potential for nutrient measurement and digestibility of Urochloa spp. grasses. In addition, the ABL method used for lignin quantification was better correlated with IVDMD and IVNDFD than the ADL method.

Diet, marker and fecal sampling method interactions with internal and external marker pairs when estimating dry matter intake in beef cattle

The use of markers is currently the most used technique for estimating feed intake in production animals when direct measurement is not possible. Twelve growing Nellore bulls were used in a crossover design aiming to evaluate the accuracy of internal markers to estimate dry matter digestibility (DMD), the accuracy of external markers to estimate fecal output (FO), and the combination of internal and external markers to estimate dry matter intake (DMI) at different fecal sampling procedures. Animals received one of the four diets which varied in forage source (corn silage or Tifton-85 hay) and forage to concentrate ratio (F:C; 60:40 or 40:60) in a 2 × 2 factorial arrangement. The internal markers, acetyl bromide lignin (ABL), indigestible aNDFom (iNDF), indigestible ADFom (iADF) and cutin, are naturally present in the feedstuffs and the external markers, Cr2O3 and TiO2, were daily fed to the animals. Three fecal sampling procedures were tested: bulk 72-h (continuous feces collection for 72 h), grab 9 × 3 (sample collection every 9 h over 3-d period), and grab 4 × 4 (4 fecal samples collected during daylight hours over 4-d period). None of the internal or external markers evaluated had complete fecal recovery (FR), being necessary to establish the FR of the markers in order to obtain correct estimates. The FR of ABL, iNDF and iADF were close to 100% when animals received a hay-based diets but close to 50% when feeding a corn silage-based diets, regardless the F:C ratio of the diet. However, ABL produced accurate DMD estimates only with both grab sampling procedures across all diets, while iNDF and iADF produced accurate DMD estimates across all sampling procedures and diets. Cutin failed to produce accurate DMD consistently. Both external markers produced accurate FO under both grab sampling procedures, except for TiO2 when the grab 4 × 4 sampling procedure was performed for animals receiving the diet with high silage proportion. In general, the grab sampling procedures yielded accurate DMD, FO and DMI estimates, which were similar or better than bulk 72 h sampling procedure. The combination of internal and external markers to estimate DMI produced satisfactory and accurate results, particularly when Cr2O3 or TiO2 was paired with iNDF or iADF under both grab sampling procedures. The grab sampling procedures can replace TFC, opening new possibilities for collectively housed animals.

The acetyl bromide lignin method accurately quantitates lignin in forage

The acetyl bromide lignin (ABL) has been shown to provide a similar relationship between lignin concentration and fiber degradability for grasses and legumes. In contrast, the acid detergent lignin (ADL) method results in different regression equations for grasses and legumes with a larger slope for grasses. The difference may be due to solubilization and loss of lignin in the grasses during the acid detergent fiber (ADF) procedure. While ADL and ABL values for legumes were about the same, ADL values for grasses were about half of those for ABL, supporting the theory that lignin was lost in grasses in the ADL method. We estimated this loss at about 60 %, so multiplying the ADL in grasses by 2.4 would yield the actual lignin concentrations. Applying this multiplier to ADL in grasses, but not in legumes, resulted in analogous regression lines for forage degradability, that were similar to those obtained with the ABL method. The nutritional entity ABL behaves uniformly for grasses and legumes and may properly determine measures of lignin, allowing further research on the mechanisms by which lignin acts as a barrier to enzymatic degradation of forage cell wall polysaccharides, which may provide information on the potential nutritive value of specific plants for herbivores.

A Robust Method to Quantify Cell Wall Bound Phenolics in Plant Suspension Culture Cells Using Pyrolysis-Gas Chromatography/Mass Spectrometry.

The wide-scale production of renewable fuels from lignocellulosic feedstocks continues to be hampered by the natural recalcitrance of biomass. Therefore, there is a need to develop robust and reliable methods to characterize and quantify components that contribute to this recalcitrance. In this study, we utilized a method that incorporates pyrolysis with successive gas chromatography and mass spectrometry (Py-GC/MS) to assess lignification in cell suspension cultures. This method was compared with other standard techniques such as acid-catalyzed hydrolysis, acetyl bromide lignin determination, and nitrobenzene oxidation for quantification of cell wall bound phenolic compounds. We found that Py-GC/MS can be conducted with about 250 µg of tissue sample and provides biologically relevant data, which constitutes a substantial advantage when compared to the 50–300 mg of tissue needed for the other methods. We show that when combined with multivariate statistical analyses, Py-GC/MS can distinguish cell wall components of switchgrass (Panicum virgatum) suspension cultures before and after inducing lignification. The deposition of lignin precursors on uninduced cell walls included predominantly guaiacyl-based units, 71% ferulic acid, and 5.3% p-coumaric acid. Formation of the primary and partial secondary cell wall was supported by the respective ~15× and ~1.7× increases in syringyl-based and guaiacyl-based precursors, respectively, in the induced cells. Ferulic acid was decreased by half after induction. These results provide the proof-of-concept for quick and reliable cell wall compositional analyses using Py-GC/MS and could be targeted for either translational genomics or for fundamental studies focused on understanding the molecular and physiological mechanisms regulating plant cell wall production and biomass recalcitrance.

Combining loss of function of FOLYLPOLYGLUTAMATE SYNTHETASE1 and CAFFEOYL-COA 3-O-METHYLTRANSFERASE1 for lignin reduction and improved saccharification efficiency in Arabidopsis thaliana

BackgroundDownregulation of genes involved in lignin biosynthesis and related biochemical pathways has been used as a strategy to improve biofuel production. Plant C1 metabolism provides the methyl units used for the methylation reactions carried out by two methyltransferases in the lignin biosynthetic pathway: caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Mutations in these genes resulted in lower lignin levels and altered lignin compositions. Reduced lignin levels can also be achieved by mutations in the C1 pathway gene, folylpolyglutamate synthetase1 (FPGS1), in both monocotyledons and dicotyledons, indicating a link between the C1 and lignin biosynthetic pathways. To test if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway, fpgs1ccoaomt1 double mutants were generated and functionally characterized.ResultsDouble fpgs1ccoaomt1 mutants had lower thioacidolysis lignin monomer yield and acetyl bromide lignin content than the ccoaomt1 or fpgs1 mutants and the plants themselves displayed no obvious long-term negative growth phenotypes. Moreover, extracts from the double mutants had dramatically improved enzymatic polysaccharide hydrolysis efficiencies than the single mutants: 15.1% and 20.7% higher than ccoaomt1 and fpgs1, respectively. The reduced lignin and improved sugar release of fpgs1ccoaomt1 was coupled with changes in cell-wall composition, metabolite profiles, and changes in expression of genes involved in cell-wall and lignin biosynthesis.ConclusionOur observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways. These improvements in sugar accessibility were achieved without introducing unwanted long-term plant growth and developmental defects.

Structural features of alternative lignin monomers associated with improved digestibility of artificially lignified maize cell walls

Plant biologists are seeking new approaches for modifying lignin to improve the digestion and utilization of structural polysaccharides in crop cultivars for the production of biofuels, biochemicals, and livestock. To identify promising targets for lignin bioengineering, we artificially lignified maize (Zea mays L.) cell walls with normal monolignols plus 21 structurally diverse alternative monomers to assess their suitability for lignification and for improving fiber digestibility. Lignin formation and structure were assessed by mass balance, Klason lignin, acetyl bromide lignin, gel-state 2D-NMR and thioacidolysis procedures, and digestibility was evaluated with rumen microflora and from glucose production by fungal enzymes following mild acid or base pretreatments. Highly acidic or hydrophilic monomers proved unsuitable for lignin modification because they severely depressed cell wall lignification. By contrast, monomers designed to moderately alter hydrophobicity or introduce cleavable acetal, amide, or ester functionalities into the polymer often readily formed lignin, but most failed to improve digestibility, even after chemical pretreatment. Fortunately, several types of phenylpropanoid derivatives containing multiple ester-linked catechol or pyrogallol units were identified as desirable genetic engineering targets because they readily formed wall-bound polymers and improved digestibility, presumably by blocking cross-linking of lignin to structural polysaccharides and promoting lignin fragmentation during mild acidic and especially alkaline pretreatment.

Evaluating internal and external markers versus fecal sampling procedure interactions when estimating intake in dairy cows consuming a corn silage-based diet

Feed intake assessment is a valuable tool for herd management decisions. The use of markers, either internal or external, is currently the most used technique for estimating feed intake in production animals. The experiment used 10 multiparous Holstein cows fed a corn silage-based diet, with 55:45 forage-to-concentrate ratio, the average fecal recovery (FR) of TiO2 was higher than FR of Cr2O3, and both FR were more than unity. With internal markers, acetyl bromide lignin and cutin FR were lower than unity, and average FR for indigestible neutral detergent fiber (iNDF) and indigestible acid detergent fiber (iADF) was 1.5. The FR was unaffected by the fecal sampling procedure and appears to be an intrinsic property of each molecule and how it interacts with digesta. Of the 2 external markers, only Cr2O3 produced accurate fecal output (FO) estimates and the same happened to dry matter digestibility (DMD) when iNDF and iADF were used. Estimates for DMD and FO were affected by sampling procedure; 72-h bulk [sub-sample from total feces collection (TFC)] sampling consistently produced accurate results. The grab (sub-samples taken at specific times during the day) sampling procedures were accurate when using either of the indigestible fibers (iNDF or iADF) to estimate DMD. However, grab sampling procedures can only be recommended when concomitant TFC is performed on at least one animal per treatment to determine FR. Under these conditions, Cr2O3 is a suitable marker for estimating FO, and iNDF and iADF are adequate for estimating DMD. Moreover, the Cr2O3+iADF marker pair produces accurate dry matter intake estimates and deserves further attention in ruminant nutrition studies. The method of dosing the external markers is extremely important and greatly affects and determines results. Whichever the method, it must allow the animals to display normal feeding behavior and not affect performance. The grab sampling procedures can replace TFC (once FR is established), which may open new possibilities for pasture-based or collectively housed animals.

Comparative study of some analytical methods to quantify lignin concentration in tropical grasses.

ObjectiveLignin plays a relevant role in the inhibition of cell wall (CW) structural carbohydrate degradation. Thus, obtaining accurate estimates of the lignin content in tropical plants is important in order to properly characterize the mechanism of lignin action on CW degradation. Comparing conflicting results between the different methods available for commercial use will bring insight on the subject. This way, providing data to better understand the relationship between lignin concentration and implications with tropical forage degradation.MethodsFive grass species, Brachiaria brizantha cv Marandú, Brachiaria brizantha cv Xaraés (MG-5), Panicum maximum cv Mombaça, Pennisetum purpureum cv Cameroon, and Pennisetum purpureum cv Napier, were harvested at five maturity stages. Acid detergent lignin (ADL), Klason lignin (KL), acetyl bromide lignin (ABL), and permanganate lignin (PerL) were measured on all species. Lignin concentration was correlated with in vitro degradability.ResultsHighly significant effects for maturity, lignin method and their interaction on lignin content were observed. The ADL, KL and ABL methods had similar negative correlations with degradability. The PerL method failed to reliably estimate the degradability of tropical grasses, possibly due to interference of other substances potentially soluble in the KMnO4 solution.ConclusionADL and KL methods use strong acid (H2SO4) and require determination of ash and N content in the lignin residues, therefore, increasing time and cost of analysis. The ABL method has no need for such corrections and is a fast and a convenient method for determination of total lignin content in plants, thus, it may be a good option for routine laboratory analysis.

Impact of Different Lignin Fractions on Saccharification Efficiency in Diverse Species of the Bioenergy Crop Miscanthus

Lignin is a key factor limiting saccharification of lignocellulosic feedstocks. In this comparative study, various lignin methods—including acetyl bromide lignin (ABL), acid detergent lignin (ADL), Klason lignin (KL), and modified ADL and KL determination methods—were evaluated for their potential to assess saccharification efficiency. Six diverse accessions of the bioenergy crop miscanthus were used for this analysis, which included accessions of Miscanthus sinensis, Miscanthus sacchariflorus, and hybrid species. Accessions showed large variation in lignin content. Lignin estimates were different between methods, but (highly) correlated to each other (0.54 ≤ r ≤ 0.94). The strength of negative correlations to saccharification efficiency following either alkaline or dilute acid pretreatment differed between lignin estimates. The strongest and most consistent correlations (−0.48 ≤ r ≤ −0.85) were obtained with a modified Klason lignin method. This method is suitable for high throughput analysis and was the most effective in detecting differences in lignin content (p < 0.001) between accessions.

Comparison of acetyl bromide lignin with acid detergent lignin and Klason lignin and correlation with in vitro forage degradability

The spectroscopic acetyl bromide lignin (ABL) and two versions of the gravimetric sulfuric acid lignin method [Lignin (sa)] were compared for their correlations with in vitro forage dry matter (IVDMD) and neutral detergent fiber degradability (IVNDFD) assays of 73 grass and legume samples. The two versions of Lignin (sa) were the acid detergent lignin (ADL) and Klason lignin (KL) methods. ABL and KL methods employ crude cell wall as the fibrous preparation while ADL uses acid detergent fiber. In vitro forage measures of degradability were negatively correlated with almost all lignin values, but the ABL method had the highest correlation coefficients for both grasses and legumes. Regression curves of ADL values with grass and legume IVDMD and IVNDF revealed different slopes, with steeper curves for grasses. With KL, grass and legume IVDMD and IVNDFD slopes were statistically similar and parallel. The results observed in the ADL and KL methods testify to the distance between the lines and data points, that is, these methods are poor predictors of NDF degradability. Grass and legume samples assayed with the ABL procedure, exhibited similar slopes, with parallel lines for both IVDMD and IVNDFD assays. Steeper inclination of curve for grasses relative to legumes in the ADL method may be attributed to partial loss of lignin during the procedure. This shows that ABL can accurately and consistently measure the inhibitory effect of lignin on degradation of structural carbohydrates of both grasses and legumes. The ABL method provides an accurate, easy and fast procedure for quantifying lignin in plant materials. A step-by-step laboratory procedure of the ABL method is described.

Lignification of ray parenchyma cells in the xylem of Pinus densiflora. Part I: Microscopic investigation by POM, UV microscopy, and TOF-SIMS

Abstract The lignification process from sapwood (sW) to heartwood (hW) in ray parenchyma cells (Pray) of Pinus densiflora has been analyzed by means of ultraviolet (UV) microscopy, acetyl bromide (CH3COBr) lignin determination, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The cell wall layers were localized by polarized optical microscopy (POM). POM revealed that Pray have almost no secondary wall in sW and have only the outer layer of secondary wall (S1) in the transition zone (TZ) and hW. UV microscopic observations indicated that the secondary wall of Pray, which is in contact with ray tracheids (Trray), begins to lignify in sW, while the secondary wall of Pray, which is not in contact with Trray, is partially lignified in the TZ. The secondary wall of both types of Pray is completely lignified in hW. The CH3COBr lignin content in sW is slightly lower than that in hW. In the TOF-SIMS measurements, the relative intensities of the secondary ions of guaiacyl-lignin (G-lignin) in the rays in sW are significantly lower than those in hW.

Use of Lignin Extracted from Different Plant Sources as Standards in the Spectrophotometric Acetyl Bromide Lignin Method

A nongravimetric acetyl bromide lignin (ABL) method was evaluated to quantify lignin concentration in a variety of plant materials. The traditional approach to lignin quantification required extraction of lignin with acidic dioxane and its isolation from each plant sample to construct a standard curve via spectrophotometric analysis. Lignin concentration was then measured in pre-extracted plant cell walls. However, this presented a methodological complexity because extraction and isolation procedures are lengthy and tedious, particularly if there are many samples involved. This work was targeted to simplify lignin quantification. Our hypothesis was that any lignin, regardless of its botanical origin, could be used to construct a standard curve for the purpose of determining lignin concentration in a variety of plants. To test our hypothesis, lignins were isolated from a range of diverse plants and, along with three commercial lignins, standard curves were built and compared among them. Slopes and intercepts derived from these standard curves were close enough to allow utilization of a mean extinction coefficient in the regression equation to estimate lignin concentration in any plant, independent of its botanical origin. Lignin quantification by use of a common regression equation obviates the steps of lignin extraction, isolation, and standard curve construction, which substantially expedites the ABL method. Acetyl bromide lignin method is a fast, convenient analytical procedure that may routinely be used to quantify lignin.

Enhancing exterior performance of clear coatings through photostabilization of wooden surfaces. Part 1: Treatment and characterization

The aim of this work was to produce a less photodegradation-prone substrate for clear coating by partially delignifying the surface cells of Pinus radiata boards to a depth of 2–3 mm while maintaining the integrity of the wood surface tissues in the delignified zone. To achieve this, several surface oxidative delignification treatments were trialled in the method development process and peracetic acid was chosen as the method for refinement and deployment. The treatment method was optimized to yield a significant degree of delignification compatible with the aim of producing a photostabilized yet intact wooden surface. A preweathering technique was also used as a second delignification method for producing photostabilized boards. Microscopic and chemical techniques were used to characterize the effects of chemical and preweathering treatments, which produced delignified surface envelopes 2–3 mm and 100 μm deep, respectively. Acetyl bromide lignin analyses, infrared spectroscopic analyses, and density changes of 30-μm-thick sections of peracetic-acid-treated samples, as a function of depth from the wood surface, suggested that a partial delignification had occurred that diminished with depth. Light and transmission electron microscopy provided evidence of delignification at the cellular level. In the surface layers of peracetic-acid-treated boards, all cell wall regions were delignified, with the middle lamella being the most severely affected. Lignin appeared to be completely removed from the cell corner middle lamella regions, but tracheids were still joined in other parts of the middle lamella. The S1, S2, and S3 walls were also delignified. In subsurface layers, cell walls were only partially delignified and the tissues held their integrity. In contrast, in the preweathered boards, cell walls in the outermost layers were completely separated at the middle lamella from photodegradation. Preferential lignin removal over that of hemicelluloses was achieved via oxidative treatment of solid wood. The outcome of this was the successful fulfillment of our aim to produce partially delignified wooden surfaces that retained sufficient strength and aesthetic appearance and were suitable for application in wooden structures, provided that the necessary protection measures were carried out. Due to the narrow surface zone produced, machining of such surfaces would not be recommended as the most delignified zone would be removed first. In Part 2 of this series, the photostability of treated boards, which had been clear coated and weathered, will be considered.

Characterization of 25 tropical hardwoods with Fourier transform infrared, ultraviolet resonance Raman, and 13C‐NMR cross‐polarization/magic‐angle spinning spectroscopy

AbstractTwenty‐five tropical hardwoods from Ghana were examined with Fourier transform infrared (FTIR), ultraviolet resonance Raman (UVRR), and 13C‐NMR cross‐polarization/magic‐angle spinning spectroscopy. FTIR and UVRR spectral data were subjected to principal component analysis, whereas four selected samples exhibiting large structural and compositional variation were investigated in more detail by NMR spectroscopy. The acetyl bromide lignin and α‐cellulose contents of the samples and the amount of acetone‐soluble substances were also determined. The most prominent principal components of the FTIR spectral model separated samples mainly due to the variations in lignin structures and carbohydrate contents and the amount and type of carbonyl structures, whereas UVRR spectral data distinguished between various aromatic and other unsaturated structures. The presence of condensed lignin/tannin‐type structures were detected from UVRR and FTIR loading line plots of the minor principal components. Condensed tannins were also observed in the dipolar dephasing NMR spectra. Furthermore, the NMR results showed variations in the amount of crystalline cellulose and hemicelluloses and in the syringyl/guaiacyl proportions in lignin. Both NMR and FTIR data suggest that in most cases, a higher amount of guaiacyl units correlated with higher levels of acetyl bromide lignin and better bioresistance of the samples. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 810–819, 2006

Can Lignin Be Accurately Measured?

Forages serve an important role in providing nutrients to ruminants while providing positive benefits to the environment. Forage cell wall digestibility is incomplete because of several structural features within the wall, but digestion is mostly inversely correlated with the amount of lignification that has occurred during cell wall development. Lignin is a hydrophobic polymer formed through enzyme‐mediated radical coupling of monolignols, mainly coniferyl and sinapyl alcohols. The polymer is highly resistant to degradation and generally passes through the ruminant unmodified. Though lignin is resistant to degradation, it is not easily quantified within various types of forages. Numerous methods have been developed over the years to measure lignin levels in different plant species. Most frequently used among workers involved with forage development or utilization are the acid detergent, Klason, and permanganate lignin methods. More recently, acetyl bromide has received attention as a possible lignin determination method. The acetyl bromide method is dependent on determining the absorbance of the extract in which all the lignin of a sample has been dissolved. Each of these methods gives different lignin values for the same type of forage sample. For example, acid detergent, Klason, permanganate, and acetyl bromide lignin methods give quite different values for alfalfa stems: 93, 145, 158, and 135 g lignin kg−1 cell wall, respectively. These differences can be even greater for grasses: 25, 77, 45, and 92 g kg−1 cell wall from corn (Zea mays L.) stalks analyzed by acid detergent, Klason, permanganate, and acetyl bromide lignin methods, respectively. This paper will discuss the different lignin determination methods and highlight the advantages and disadvantages of each as they relate to forage sample analysis.

Estimation and comparison of acid detergent lignin and acetyl bromide lignin in fallen leaves using near-infrared spectroscopy

Several analytical methods have been developed to measure the lignin content corresponding to different plant species and different regions. The sulphuric acid method is commonly used for objective determinations of lignin content using near-infrared spectroscopy. Lignin is a complex polymer of lignin units. The types and ratios of lignin units vary among taxonomic classes of plants. To compare the lignin content as determined by different methods of chemical analysis, fallen leaves of different species were analysed using both the acid detergent and acetyl bromide procedures. Near-infrared reflectance spectra were obtained for each sample of dried ground leaves, and stepwise multiple linear regression analyses were performed to compare the amounts of lignin determined using acid detergent and acetyl bromide. In monocotyledonous herbaceous plants, the lignin content determined by acetyl bromide was more than twice that determined by acid detergent. Despite the difference in the values, regression analysis provided acceptable results for both lignin preparations. Although the acid detergent procedure has generally been regarded as accurate for lignin determination, our results suggest that caution is required in the selection of the method of chemical analysis when using near-infrared spectroscopy to estimate the lignin content of different taxonomic classes of plants.

Hot alkali-labile linkages in the walls of the forage grass Phalaris aquatica and Lolium perenne and their relation to in vitro wall digestibility

The factors affecting in vitro dry matter digestibility (IVDMD) of fully mature internodes of 150 lines of the forage grass, Phalaris aquatica, and internodes of 100 lines of perennial ryegrass ( Lolium perenne), harvested just after anthesis, were investigated. The relationships between IVDMD and the contents of acetyl bromide lignin, and ester–ether linkages between lignin and wall polysaccharides, measured by hydroxycinnamic acids (HCAs) released by 4 M NaOH at 170 °C respectively, were determined. The regression analysis gave r 2=0.05 and 0.03 for the relation between IVDMD and lignin content and r 2=0.51 and 0.53 for the relation between IVDMD and the content of hot alkali-labile HCA (predominantly ferulic acid) for phalaris and ryegrass, respectively. These observations are interpreted in terms of the restricted accessibility of polysaccharide hydrolysing enzymes to their substrates in the forage cell walls by the covalent cross-linking of wall polymers through HCAs.

Rendimento e comportamento espectrofotométrico da lignina extraída de preparações de parede celular, fibra em detergente neutro ou fibra em detergente ácido

A lignina extraída da planta forrageira pelo reagente brometo de acetila (lignina brometo de acetila - LBrAc), para ser empregada como padrão de referência no método espectrofotométrico para a determinação quantitativa da lignina, não pode originar-se da planta intacta, devido à interferência de diversas outras substâncias, que absorvem no mesmo comprimento de onda que a lignina; aconselha-se, portanto, que a extração da lignina seja realizada a partir de um dos seguintes compostos: parede celular propriamente dita (PC), fibra em detergente neutro (FDN) ou fibra em detergente ácido (FDA). Os rendimentos das extrações das ligninas, bem como o comportamento espectrofotométrico das mesmas, foram avaliados nestes três compostos provenientes de diversas plantas forrageiras. Na média, os rendimentos das extrações de LBrAc foram relativamente uniformes entre si, entretanto, como a concentração de FDA foi a menor entre todas as preparações (devido principalmente à solubilização da hemicelulose) e como esta não apresentou maior capacidade extrativa da lignina, o rendimento de LBrAc proveniente da FDA, com base na planta inteira, foi o menor observado. A absorção da luz ultravioleta das diversas ligninas foi desigual, revelada pelos espectrogramas traçados, de tal modo a sugerir que a preparação de parede celular adotada para o isolamento da lignina deve ser a mesma para a determinação final da concentração de lignina.

Changes in thioacidolysis products of lignin in wheat straw as affected by SO 2 treatment and passage through the gastro-intestine of sheep

Sheep were fed two rations based on untreated (WS) and SO 2-treated (SO 2–WS) wheat straw. The neutral detergent fiber (NDF) preparations and water soluble lignins (WSL) isolated from straws, rumen liquor and feces were subjected to thioacidolysis, to study the effect of treatment and passage through the gastro-intestine on monolignol composition of the lignins. SO 2 treatment affected guaiacyl ( G) and syringyl ( S) contents of the straw cell walls with major effect on S, reducing G to 66% and S to 26% of their original concentrations, thus changing the S/ G ratio in the CW fraction from 1.6 in WS to 0.64 in SO 2–WS. Concomitantly, as a reflection of the above-mentioned there was an increase in the S/ G ratio of the WSL, from 0.54 in WS to 1.27 in SO 2–WS. Fecal CW residues were richer in p-hydroxyphenyl (H), G and S as compared with the original straw cell walls; the gap being particularly large in the SO 2–WS treatment in which the concentrations of monolignols in CW residues were increased by more than ten-fold. The concentration of WSL in the rumen of sheep fed the SO 2–WS ration was about eight-fold that found in the rumen of the WS sheep, most likely a result of the combined chemical (SO 2) and digestive (rumen microorganisms) effects. Most G and S were found in the CW fraction of the untreated straw, whereas in the SO 2-treated straw, the majority of G and S were in the water soluble lignin fraction. In the untreated wheat straw ration (WS), 25.7% of G and 36.6% of S disappeared from the digestive tract. Feed CW- G was higher by 18% and feed CW- S by 60% than the respective fecal CW components in the WS treatment, accompanied by only modest quantitative changes between feed and feces, in the G and S components of WSL. In the SO 2–WS treatment, CW- G was increased from 3.48 to 6.26 g/day (by 80%) and CW- S from 2.58 to 6.56 g/day (by 154%) between feed and feces. The digestibility of total acetyl bromide lignin (ABL) was in accord with that of the monolignol data namely, a small proportion (11.4%) of total ABL disappeared from the gastro-intestine of the WS sheep, but a minor, almost negligible percentage (2.94%) of the ABL was digested by the SO 2–WS sheep.