Luanne L. Rigsby

Chemical and structural analysis of fibre and core tissues from flax

Samples of flax (Linum usitatissimum) stems from the cultivars ‘Natasja’ and ‘Ariane’ were separated into fibre and core fractions and analysed by gas–liquid chromatographic methods, 13C CPMAS NMR spectrometry, histochemistry, electron microscopy and UV absorption microspectrophotometry to assist in determining the structure and composition of these cell walls in relation to quality and utilisation. Analyses from chromatography and NMR gave similar results for carbohydrate and phenolic constituents in various samples and in the lower, more mature regions of the stem. Amounts of uronic acids and xylose were lower while amounts of mannose, galactose and glucose were higher in fibre vs core fractions. Quantities of phenolic constituents were significantly higher in the core than the fibre, with groups representative of both guaiacyl and syringyl lignins; amounts of phenolic acids were low. NMR showed a low intensity signal for aromatics in fibre, and it is possible that such signals arise from compounds in the cuticle rather than the fibre. Microscopic studies indicated that aromatic constituents were present in core cell walls, cuticle of the epidermis, and cell corners and middle lamellae of some regions within the fibre tissues. The lignin in fibre appeared to be of the guaiacyl type and may be too low in concentration to be unambiguously detected by NMR. Aromatic compounds were not observed in the epidermis or parenchyma cell walls. Similar analyses of dew-retted (unscutched) samples indicated that core tissues were mostly unchanged from unretted samples. Retted fibre tissues still contained lignified cell corners and middle lamellae in some regions. The cuticle, which was associated with retted fibres, was not degraded by dew-retting fungi. Fungi removed interfibre materials in some places and at times degraded the secondary wall near the cell lumen of fibre cells. Results indicate that microspectrophotometry and histochemistry are useful to identify the location and type of aromatics in fibre cell walls.

Influence of Chelating Agents and Mechanical Pretreatment on Enzymatic Retting of Flax

Adding chelating agents, i.e., oxalic acid and ethylenediamine-tetra-acetic acid (edta), substantially increases the retting effect on flax by the commercial enzyme products Ultrazym and Flaxzyme (Novo Nordisk), as shown by scanning electron microscopy, release of reducing sugars, and the Fried test. Degradation of pectin-rich citrus peel by these enzymes also increases with the addition of oxalic acid and edta, while citric acid has a low or insignificant effect. Oxalic acid at 50 mmol concentration reduces the amount of Flaxzyme required to effectively ret flax stems, according to the Fried test, by a factor of about 50. Retting with Flaxzyme and 50 mmol oxalic acid is completed in approximately half the time at 45°C, compared with that at 22°C. A mechanical pretreatment that crushes flax stems by pulling them over a surface at a 90° angle opens the flax structure and further increases the efficiency of enzymatic retting. These procedures appear to modify both the chemical and structural features of flax, and they reduce the time as well as the amount of enzyme required to ret flax, therefore improving technical efficiency and economic attractiveness at the commercial level.

Effect of Retting Enzymes on the Structure and Composition of Flax Cell Walls

Commercial enzyme mixtures are tested for their possibly selective degradation of flax (Linum usitatissimum L.) stem components in relation to the retting process in producing linen. Structural and chemical compositional results from treatments are obtained using scanning electron microscopy, histochemistry, gas-liquid chromatography, 13C cp mas nmr spectrometry, and mid-infrared spectroscopy. Flaxzyme and Ultrazym and an enriched pectinase mixture (epm), which was not developed for flax retting but is included for comparison, are tested for their activity toward cell wall components and used in various concentrations for “enzyme-retting” of flax. Ariane flax stem sections are incubated with enzymes in a rotary incubator and the fibers are manually separated from the residual core. All of the commercial enzyme mixtures have cellulase, pectinase, and hemicellulase activities, but individual enzyme activities vary. Activities against the soluble test substrates do not predict the activity against natural fibers. At about equal protein concentrations, Flaxzyme treatment appears to facilitate bast fiber removal better than the other enzymes, with Ultrazym nearly as effective and epm the least effective. The ranking of effectiveness is generally supported by the amounts of uronic acid, arabinose, and xylose removed from the stems analyzed chemically. Increased enzyme levels generally facilitate removal of matrix carbohydrates from the flax. All enzymes separate bast fibers from the lignified core and partially from the cuticle near the cut surface of the stem sections, but the enzymes do not work far from the exposed ends. Retting quality is defined more by the degree of cell wall degradation and fiber separation than by any differences in kinds of cell walls degraded by the various enzymes. The cuticle remains attached to the fiber at times, apparently reducing access of the enzymes to the matrix polysacchrides and suggesting some recalcitrance of epidermal cells (and therefore loss of cuticle) to biodegradation. Lignin remains in the middle lamellae after enzyme retting and would likely prevent separation of the fiber bundles. Some solubilzation of the inner secondary wall of the flax fiber appears to occur with Flaxzyme. The structural and chemical analyses characterize alterations in flax bast after enzyme retting and would be useful in ranking the specificity and effectiveness of cell wall degradation.

Population changes of fibrolytic rumen bacteria in the presence of phenolic acids and plant extracts

Abstract Phenolic acids inhibit rumen microbial digestion of forage, but information on the interactions of the mixed population with natural phenolics from plants is required to elucidate fully the anti-quality aspect of phenolic compounds in forages. The objective of this study was to evaluate the digestion of leaf blades of Coastal bermudagrass ( Cynodon dactylon L. Pers.) and Italian ryegrass cv. RvP ( Lolium multiflorum L.) and the morphological types of colonizing bacteria during incubation with rumen fluid in the presence of phenolic acids [ trans - p -counmaric acid (t-PCA), cis - p -coumaric acid, trans -ferulic acid, or diferulic acid] or phenolic extracts from maize ( Zea mays L.) stems or barley ( Hordeum vulgare L.) straw. The presence of phenolics resulted in less degradation of plant cell walls, as determined by scanning electron microscopy and often fewer bacteria associated with the plant walls, as determined by transmission electron microscopy, when compared with control treatments. In the presence of t-PCA, the ability of microbial suspensions that had not been previously exposed to t-PCA to colonize and degrade leaf blade cell walls was completely inhibited. This inhibitory effect was reduced, but not totally eliminated, if microbial suspensions were exposed to t-PCA for 48 h in culture prior to inoculation of leaf blades. Trans -PCA and phenolics from maize stem caused a reduction in the proportion of fiber-associated bacteria resembling Bacteroides succinogenes , which appeared to be the most active fiber digester in this system. Phenolics extracted from cell walls of barley straw showed slight to no inhibition and were similar to control treatments.

Botanical fractions of rice straw colonized by white-rot fungi: changes in chemical composition and structure

Three species of white-rot fungi (Cyathus stercoreus (Cs) ATCC-36910, Phanerochaete chrysosporium (Pc) BKM, and Pleurotus sajor-caju (Ps) 537) were grown on leaf blade (leaf) or stem plus leaf sheath (stem) of rice straw for 30 d by solid state fermentation (SSF). Physical and chemical methods were employed to evaluate substrate specificity, substrate composition and histology. Changes in histology of decayed material were evaluated before and after ruminal digestion by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Control leaf and stem were similar in IVDMD (38%), although leaf was higher in crude protein and lower in cell wall compared to stem (3.7 vs. 2.8%; 73.9 vs. 80.7%, respectively). The changes were due mostly to a higher concentration of silica in leaf compared to stem (17.0 vs. 13.1%). After 30 d of SSF, Cs and Ps increased the IVDMD of leaf from 38.1 to 49 and 46.3%, respectively, by selective degradation of hemicellulose as opposed to cellulose. In contrast, Pc degraded cellulose and hemicellulose indiscriminately in leaf and lowered the IVDMD of leaf to 30.1%. Partially degraded lignin, silica and hemicellulose of leaf were negatively correlated (r) with IVDMD in contrast to cellulose (r = −0.49, −0.54, −0.16 and 0.85, respectively). Prediction of IVDMD of fungal-decayed leaf was primarily a function of hemicellulose and cellulose with a coefficient of IVDMD = −0.155 + 2.14 (cellulose) −0.87 (hemicellulose); R2 = 0.98. Stem decayed by Pc and Cs became less digestible compared to the control (18.5 and 20.3% vs. 39.7%, respectively), although hemicellulose and cellulose of stem were poorly degraded after SSF. Only Ps improved the IVDMD of stem compared to the control (44.1 vs. 39.7%). SEM sections of leaf decayed by Pc showed complete degradation of mesophyll but the more recalcitrant vascular and epidermal tissues resisted rumen degradation and resulted in lower IVDMD. Leaf tissues colonized by Cs and Ps showed presence of all tissues but after 72 h rumen microorganisms completely degraded mesophyll tissue which resulted in a higher IVDMD. Observation of TEM sections showed that fungal treatment facilitated rumen microbial penetration of lignified tissues. Improvement of digestibility of decayed straw depends upon the fungal species, the plant substrates and the botanical fractions.

Quality Properties of Flax Fibers Retted with Enzymes

Flax that has been mechanically treated in an opener-blender to disrupt stem integrity is enzymatically retted with a series of enzyme formulations with different levels of Flaxzyme with or without ethylenediamine-tetra-acetic acid (EDTA) as a chelator. Samples are then characterized by light and transmission electron microscopy and fiber tests for micronaire, strength (g/tex), and elongation. Unretted control fibers are off scale for micronaire and consist of fiber bundles with associated epidermis/cuticle fragments. Structurally, the lowest level of Flaxzyme used, i.e., 0.05% (w/v), plus 50 mmol EDTA produces ultimate fibers and bundles of various sizes without evidence of associated epidermis/cuticle. This formulation yields flax fibers with similar micronaire values and about 34% more strength than the recommended level of 0.3% Flaxzyme. A level of 3.0% Flaxzyme extensively removes middle lamellae from the bundles, produces the smallest micronaire values, and reduces strength to about 30% of fibers treated with 0.05% Flaxzyme plus chelator. Variations in fiber properties with the different formulations suggest that pectin degradation varies for different regions of the bast tissues and that specific strategies for improving enzymatic retting can be developed. Results show that fiber quality tests for cotton are useful in differentiating formulations for enzymatic retting of flax, and 0.05% Flaxzyme plus 50 mmol EDTA is the most efficient retting formulation.

Biological delignification of plant components by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus

Lignocelluloses from diverse plant types were treated with the white rot fungi Ceriporiopsis subvermispora (strains CZ-3-8497 and FP-90031-sp) and Cyathus stercoreus. Sources of lignocellulose included: the warm-season grasses sorghum (leaf blades, sheaths, and stems), pearl millet, napiergrass, and maize (stems); the cool-season grass wheat (leaf blades, sheaths, and stems); the legumes alfalfa (stems) and lespedeza (leaflets and stems). Fungus-treated residues were compared with untreated, control samples and with plants treated with a non-delignifying isolate of Trichoderma. Residues were evaluated for improved biodegradability by ruminal microorganisms and modifications in cell wall chemistry by nuclear magnetic resonance, gas chromatography, and ultraviolet absorption microspectrophotometry. Specific plant—fungus interactions were identified that resulted in selective removal of lignin and improved biodegradability by white rot fungi but not the Trichoderma sp. All white rot fungi removed ester-linked p-coumaric and ferulic acids from grass stems, and this phenomenon appeared to account for the significant reduction in aromatic components and improved biodegradability of fungus-treated grass lignocellulose. Cell walls in alfalfa stems were more resistant to biological delignification than those in grasses, with only C. stercoreus removing significant amounts of aromatics and improving biodegradability. All white rot fungi improved the biodegradability of tannin-rich lespedeza samples.

Chelating Agents and Enzyme Retting of Flax

Chelators added to pectinase-rich enzyme mixtures increase the efficiency of enzyme retting of flax. The multitude of available chelators requires research to optimize enzyme retting for cost and fiber quality, Of several chelators tested, ethylenediaminetetraacetic acid (EDTA) is the most effective in sequestering calcium from solution, with substantial activity even at pH 4 or 5. Phosphate is also effective, but only at high pH. EDTA and Mayoquest 200, a commercial product with ∼37% EDTA, in combination with Viscozyme L or Lyvelin, commercial enzymes with polygalacturonase activities, yield high Fried test scores, indicating efficient separation of fibers from core tissues. Neither chelator nor enzyme at the levels tested alone effectively rets flax at a pH of around 5. In contrast, EDTA at 20 mmol/L levels and alkaline pH yields high Fried test scores. The addition of BioPrep, a commercial pectate lyase, does not influence retting measured by the Fried test with EDTA at alkaline pH, but the enzyme does improve retting with the weaker chelator sodium tripolyphosphate. The quality of fibers, as determined by the yield of fine fibers obtained by passing retted flax through the Shirley analyzer, is substantially greater with EDTA plus Viscozyme at pH 5 than alkaline chemical retting with EDTA, sodium tri polyphosphate, or sodium oxalate without enzyme. EDTA is the most effective chelator at acidic pH levels for stimulating flax retting by various commercial pectinase-rich enzyme mixtures. However, other less expensive candidates require additional study for more cost-effective formulations and applicable fiber properties.

Influence of water presoak on enzyme-retting of flax

Abstract Enzyme-retting offers an alternative to the current method of dew-retting to extract fibers from flax (Linum usitatissimum L.). Additional steps could improve the efficiency of enzyme-retting and modify the properties of the resulting fibers. Samples of ‘Ariane’ flax, which were grown in South Carolina during the winter and harvested early for quality fiber or late for both fiber and seed, were presoaked with distilled water before enzyme-retting. Soaked, enzyme-retted, and air-dried fibers were compared with unsoaked, control samples for yield and properties, and the water extract (or a freeze-dried portion) was tested in various methods for its influence on enzyme-retting. Presoaking increased fine fiber yield in some cases, but fiber strength at times was reduced. Analyses of the freeze-dried residue from soaking showed a mixture of sugars (128.6 and 101 mg g−1 for early and late harvest, respectively) and aromatic components including p-coumaric and ferulic acids and guaiacyl and syringyl units (3.51 and 3.05 mg g−1 total aromatics for early and late harvest, respectively). Water extracts from presoaking treatments at 1.0–2.0% (w/v) were not inhibitory to the retting fungus Rhizopus oryzae sb or to Viscozyme used for enzyme-retting, based on the Fried test and enzyme activities. Turbidity tests showed slight growth inhibition for Eschericia coli and Streptococcus sp. in the presence of water extracts from early versus late harvest flax at 0.5% (w/v), with those from late harvest flax more inhibitory. Benefits on the efficiency of water presoaking prior to enzyme-retting were moderate and not uniform in this study, and modifications may depend upon particular flax harvests.

Retting Flax with Endopolygalacturonase from Rhizopus oryzae

Retting, which is the process of separating fibers from nonfiber tissues in bast plants, is the major problem in processing flax for linen, and new methods, especially enzyme retting, are being pursued to overcome this problem. Production of an endopolygalact uronase (EPG) from Rhizopus oryzae, which was isolated earllir from dew retted flax, is optimized, and the enzyme is subsequently purified and characterized. Purified EPG is evaluated for its efficiency in retting flax alone and in combination with other cell wall degrading enzymes. The Fried test, light and scanning electron microscopy, and fiber strength and fineness properties indicate that EPG alone (without additional enzymes) plus chelator gives retting efficiencies similar to previously used enzyme mixtures. The addition to EPG of pectin methyl esterase, pectin lyase. xylanase, or endoglucanase does not improve retting efficiency or strength and fineness properties of the retted flax fibers over EPG alone. Spray enzyme retting of a 50 g flax sample with EPG/chelator formulation produces retted flax fibers with strength and fineness properties similar to those retted with a commercial enzyme mixture. Our results indicate that EPG is of paramount importance in flax retting, and they suggest an opportunity, through cloning technology, to produce a consistently effective but simplified enzyme formulation for flax retting.