Biopreparation of cotton fabric with enzymes produced by solid-state fermentation

Fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens, produced by solid-state fermentation (SSF) on cotton seed-coat fragment waste as substrate and enzyme inducer were investigated in biopreparation of cotton fabric. Cotton seed-coat fragment is rich in lignin, cellulose and hemicelluloses, therefore enzyme complexes produced by target fungi on such a substrate can be used effectively to degrade impurities in cotton fabrics during biopreparation. Activities of extracellular hydrolytic and ligninolytic enzymes were determined from the SSF extract materials. The potential of the hydrolytic and accompanying oxidative enzymes in the whole SSF cultures was exploited in degradation of seed-coat fragments and other coloring materials of greige cotton fabric. Enzyme assays indicated that many extracellular enzymes have been produced under these conditions including both hydrolytic and oxidative enzymes. A. oryzae NRRL 3485 produced significantly higher amounts of both hydrolytic and oxidative enzymes than other tested fungi. Best results in removal of seed-coat fragments from cotton fabric were obtained by P. chrysosporium NCAIM (=ATCC 34541), P. chrysosporium VKM F-1767 and A. oryzae NRRL 3485 SSF enzyme complexes.     

Enzymes and chelating agent in cotton pretreatment

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase–pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.     

Notes on the ontogeny and structure of the seed-coat of Sclerocarya birrea (Richard) Hochst. subsp. caffra (Sonder) Kokwaro (Anacardiaceae)

The pendulous, bitegmic, anatropous ovulr with dorsal raphe is suspended at the tip of a massive funicle. A group of nurellar cells with intensively staining cell walls, the hypostase sensu stricto , is present. The initially plate-like tanniniferous chalazal-nucellar tissue, with suberin and lignin impregnated cell walls represents a hypostase sensu lato . The mature seed-coat is formed by the raphe, extensive chalaza, adjacent, well-developed, cup-like hypostase sensu lato , remnants of the two integuments and a cuticular layer. The exalbuminous seed of Sclerocarya birrea suhsp. caffra (the Marula), is regarded to he a derived and phylogenetically advanced type. The undifferentiated seed-roat is very similar to that found in Lannea discolor which, like the marula, belongs to the tribe Spondieae. The similarities in the structure of the seed-coat and seed of the marula and L. discolor confirm their proposed close phylogenetir relationship.     

Seed-coat anatomy of japanese species ofCorydalis andDicentra (Papaveraceae; Fumarioideae)

Development and structure of seed-coat were examined in 16 species ofCorydalis and two species ofDicentra. Neither the tegmen (developed ii) nor the testa (developed oi) is multiplicative during seed development. Mature seed-coat is consistent in all the species examined in having mechanical structure in the exo- or endotesta. Differences are found in (1) which of the integumentary layers develops into the main mechanical layer (exotesta or endotesta), (2) whether a mesotesta is differentiated or not, and (3) whether the endotegmen is persistent as a layer of thick-walled cells, is persistent only partially, or is thoroughly degenerated. Theses seed-coat characters distinguish six groups of species (i.e., four groups inCorydalis and two inDicentra), which represent well infrageneric taxa proposed on the other characters. Evidence from seed-coat anatomy further suggests thatDicentra spectabilis (subg.Hedycapnos) retains the primitive (endotestal) seed-coat of Fumarioideae.     

Steam explosion lignins; their extraction,structure and potential as feedstock for biodiesel and chemicals

In the present study, a steam explosion wood pre-treatment process, optimized earlier with respect to ethanol production, has been applied to both softwoods (Picea abies and Pinus sylvestris) and hardwoods (Betula verrucosa and Populus tremula). The alkaline extractable lignins have then been isolated to investigate lignin separation efficiency and lignin structure and to evaluate their potential for producing value-added products, such as biodiesel components or chemicals, in terms of the purity, molecular size, functional groups, β-O-4′ inter-unit linkage content, and degradability in a subsequent processing treatment. The mechanism of lignin modification and possible improvements to the steam explosion pre-treatment process are discussed.     

Effects of thermo-chemical pre-treatment on anaerobic biodegradability and hydrolysis of lignocellulosic biomass

The effects of different thermo-chemical pre-treatment methods were determined on the biodegradability and hydrolysis rate of lignocellulosic biomass. Three plant species, hay, straw and bracken were thermo-chemically pre-treated with calcium hydroxide, ammonium carbonate and maleic acid. After pre-treatment, the plant material was anaerobically digested in batch bottles under mesophilic conditions for 40 days. From the pre-treatment and subsequent anaerobic digestion experiments, it was concluded that when the lignin content of the plant material is high, thermo-chemical pre-treatments have a positive effect on the biodegradability of the substrate. Calcium hydroxide pre-treatment improves the biodegradability of lignocellulosic biomass, especially for high lignin content substrates, like bracken. Maleic acid generates the highest percentage of dissolved COD during pre-treatment. Ammonium pre-treatment only showed a clear effect on biodegradability for straw.     

Development and structure of the seed-coat of Lannea discolor (Sonder) Engl. (Anacardiaceae)

VON TEICHMAN, I., 1988. The development and structure of the seed-coat of Lannea discolor (Sonder) Engl. (Anacardiaceae). The bitegmic, anatropous ovule contains a group of nucellar cells with slightly thickened and intensively staining cell walls. Besides this hypostase sensu stricto, the nucellus cells in the chalaza become tanniniferous. This tanniniferous chalazal-nucellar tissue is intially plate-like. It is referred to as the hypostase sensu lato. The latter and the chalaza enlarge significantly. The raphe, extensive chalaza and well-developed cup-like hypostase sensu lato play an important role in the development of the seed-coat. The inner, tanniniferous epidermis of the inner integument persists in parts of the mature seed-coat. The outer, distinctly tanniniferous epidermis of the outer integument shows in the mature seed-coat a degree of secondary wall thickening. This undifferentiated type of seed-coat of L. discolor (tribe Spondieae) is remarkably similar to that of Camnosperma minor (tribe Rhoideae), both also showing tendency towards the exotestal type. In the Rhoideae the endotestal, i.e. differentiated type, of seed-coat is also present. The exalbuminous seed of L. discolor represents a derived and advanced type.     

Changes in lignin content of leaf litters during mulching

Alkaline nitrobenzene oxidation, ozonation and methoxyl content determinations were applied to decomposing leaf litter of Ginkgo biloba L., Cinnamomum camphora sieb., Zelkova serrata Makino and Firmiana simplex W. F. Wight, respectively, during mulching to investigate the properties and estimate changes in lignin composition and content. Since the Klason lignin residue originated from components highly resistant to degradation by acid, the methoxyl content of Klason residue was used to estimate the lignin content of leaf litter. Quantitative analysis of presumed lignin-derived fragments, by use of alkaline nitrobenzene oxidation and ozonation methods, suggested that the estimated lignin content approximates that of the real lignin content of leaves, which is greatly overestimated by the Klason procedure. The estimated lignin contents ranged from 3.9 to 10.0% while the Klason lignan residue varied from 37.1 to 46.7% in un-mulched leaf litter. The absolute amounts of the measured lignin somewhat decreased during mulching, while the structure of lignin remaining in leaf litters after mulching was considered not to be very different from its original structure.     

内蒙古紫皮小麦紫皮性状的遗传分析

本文对内蒙古紫皮小麦进行了遗传分析。研究了这一材料的来源,普通小麦与内蒙古紫皮小麦杂交后代的细胞学特征,回交一代和杂种第二代的紫皮性状表现,以及内蒙古紫皮小麦、普通小麦和赖草的同工酶。综合研究的结果初步表明,这一紫皮小麦与国外报道的紫皮小麦材料不同。本材料是由普通小麦(Triticum aestivum L.)与赖草(Leymus dasystachys(Trin.) Dilger)杂交,经回交、自交后得到的,而国外报道的紫皮小麦材料是由普通六倍体小麦与四倍体紫皮小麦杂交得到的。内蒙古紫皮小麦很可能是一个异代换系,具有一对来自赖草的染色体,控制紫皮性状的基因在这对外源染色体上,其紫皮性状能稳定遗传,受控于一对显性基因。     

Intra- and Extracellular Localization of Lignin Peroxidase during the Degradation of Solid Wood and Wood Fragments by Phanerochaete chrysosporium by Using Transmission Electron Microscopy and Immuno-Gold Labeling

The distribution of lignin peroxidase during degradation of both wood and woody fragments by the white rot fungus Phanerochaete chrysosporium was investigated by using anti-lignin peroxidase in conjunction with postembedding transmission electron microscopy and immuno-gold labeling techniques. The enzyme was localized in the peripheral regions of the fungal cell cytoplasm in association with the cell membrane, fungal cell wall, and extracellular slime materials. In solid wood, lignin peroxidase was detected in low concentrations associated with both superficial and degradation zones within secondary cell walls undergoing fungal attack. A similar but much greater level of extracellular peroxidase activity was associated with wood fragments degraded by the fungus grown under liquid culture conditions optimal for production of the enzyme. Efforts to infiltrate degraded wood pieces with high levels of lignin peroxidase showed the enzyme to be restricted to superficial regions of wood decay and to penetrate wood cell walls only where the wall structure had been modified. In this respect the enzyme was able to penetrate characteristic zones of degradation within the secondary walls of fibers to sites of lignin attack. This suggests a possibility for a close substrate-enzyme association during wood cell wall degradation.     

Kinetic Modeling and Mechanisms of Acid-Catalyzed Delignification of Sugarcane Bagasse by Aqueous Acetic Acid

Organosolv pretreatment of lignocellulose pertains to a biomass fractionation process to obtain cellulosic pulp, high-purity lignin, and hemicellulosic syrup. In the present work, sugarcane bagasse was delignified by aqueous acetic acid (AcH) under atmospheric pressure with addition of sulfuric acid (SA) as a catalyst. Based on the multilayered structure of plant cell wall and the inhibitive effect of dissolved lignin on delignification rate, a novel pseudo-homogeneous kinetic model was proposed by introducing the concept of “potential degree of delignification (d _D)” into the model. It was found that delignification rate was a first-order reaction with respect to SA concentration, while AcH concentration showed a high reaction order to delignification rate. The activation energy for delignification was determined to be 64.41 kJ/mol. The relationships of kinetic constants and d _D with reaction temperature, AcH, and SA concentrations were determined according to experimental data. Mechanism analysis indicated that cleavage of α-aryl ethers bonds were mainly responsible for the formation of lignin fragments. AcH concentration affected the solubility parameter (δ value) of AcH solution and the ability to form hydrogen bonds with lignin fragments. Therefore, the driving force for solubilizing lignin fragments increased with AcH concentration, and thus AcH concentration had a very significant influence on delignification rate.     

Genetics of seed-coat colour in Citrullus lanatus (Thunb.) Mansf.

Summary Intervarietal crosses in watermelon, Citrullus lanatus (Thunb.) Mansf., involving six parents with black (J18-1 and J 75), brown (J56-1 and N.H. Midget), red (Bykovski-199) or light cream (Red Nectar) seed-coat colour were made. Parents, F₁, F₂ and backcross populations were evaluated for their phenotypic expressions with regard to the seed-coat colours involved. Black colour was monogenically dominant over brown light cream and red colour of seed-coat separately or independently. Red colour was dominant over light cream colour of seed-coat by a single pair of genes. The light cream colour was recessive to the brown seed-coat colour of watermelon where a single pair of genes was involved.     

Polymerization of lignin fragments contained in a model effluent by polyphenoloxidases and horseradish peroxidase/hydrogen peroxide system*

An effluent containing soluble lignin fragments was treated with potato-polyphenoloxidases (PPO) or horseradish peroxidase/hydrogen peroxide system (HRP/H(2)O(2)). In both cases the reaction was evidenced by the formation of a brown precipitate that was a consequence of the polymerization of lignin fragments. The effect of reaction time, pH, and amount of soluble lignin per unit of enzyme activity on the insolubilization yield was evaluated for PPO-initiated reactions. For HRP-initiated reactions, the amount of H(2)O(2) per unit of enzyme activity was also evaluated. Mathematical models were calculated to predict the insolubilization yield as a function of the significant variables. Based on these models, the insolubilization reaction was optimized and reached maximal values of ca. 50% in both reaction systems. Higher insolubilization yields were not achieved. Chemical characterization of the soluble lignin fragments indicated that the insolubilization yield could not be improved, because the lignin fragments had limited amounts of free phenolic substructures available for the initial steps of the polymerization reaction.     

Molecular weight distribution and structural characteristics of polymers obtained from acid soluble lignin treated by oxidative enzymes

Brown precipitates were obtained by polymerization of low molecular weight lignin fragments contained in a model effluent. Polymerization reactions were initiated by potato-polyphenoloxidase (PPO) or horseradish peroxidase/H(2)O(2) system (HRP/H(2)O(2)). The insolubilization processes occurred after a molecular weight increase of the lignin, as shown by gel permeation chromatography (GPC). The effect of reaction time, pH and amount of soluble lignin per unit of enzyme activity on the molecular weight distribution was evaluated for PPO-initiated reactions. For HRP-initiated system the amount of H(2)O(2) per unit of enzyme activity was also evaluated. Chemical characterization of the macromolecules obtained under optimized conditions and the soluble lignin fragments present in the effluent suggests that the polymerization reactions occur by oxidative cleavage of alpha-beta unsaturated bonds of the soluble lignin fragments. Methoxyl group analysis showed that p-hydroxycoumaryl units were preferentially oxidized by PPO. In contrast, HRP oxidized preferentially guaiacyl and siringyl units giving more condensed polymers.     

The ontogeny and structure of the pericarp and seed-coat of Harpephyllum caffrum Bernh. ex Krauss (Anacardiaceae)

The exocarp sensu lato , which develops from the outer epidermis and adjacent parenchyma of the ovary wall, consists of collenchyma cells with a stomatous epidermis. The fleshy, parenchymatous mesocarp or sarcocarp develops after endocarp differentiation. The endocarp is partly spongy and partly woody. The spongy endocarp contains most of the vascular tissue and fills the cavities and grooves of the intricately sculptured outer woody endocarp. The inner woody endocarp and adjacent woody, endocarpal operculum develop from the inner epidermis and subepidermal parenchyma of the ovary wall. The bitegmic, anatropous ovule develops into a derived, exalbuminous seed with an undifferentiated seed-coat. An extensive chalaza, extensive hypostase sensu lato and the raphe are important in the development of the seed-coat. The pericarp and seed-coat of H. caffrum is compared with those of Sclerocarya birrea subsp. caffra and Lannea discolor . The close phylogenetic relationship of these three species of the Spondieae is reaffirmed. The marked similarities in pericarp and seed structure between H. caffrum and species of the genus Spondias are noted.     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

Characterization of degradation products from alkaline wet oxidation of wheat straw

Alkaline wet oxidation pre-treatment (water, sodium carbonate, oxygen, high temperature and pressure) of wheat straw was performed as a 2(4-1) fractional factorial design with the process parameters: temperature, reaction time, sodium carbonate and oxygen. Alkaline wet oxidation was an efficient pre-treatment of wheat straw that resulted in solid fractions with high cellulose recovery (96%) and high enzymatic convertibility to glucose (67%). Carbonate and temperature were the most important factors for fractionation of wheat straw by wet oxidation. Optimal conditions were 10 min at 195 degrees C with addition of 12 bar oxygen and 6.5 g l(-1) Na2CO3. At these conditions the hemicellulose fraction from 100 g straw consisted of soluble hemicellulose (16 g), low molecular weight carboxylic acids (11 g), monomeric phenols (0.48 g) and 2-furoic acid (0.01 g). Formic acid and acetic acid constituted the majority of degradation products (8.5 g). The main phenol monomers were 4-hydroxybenzaldehyde, vanillin, syringaldehyde. acetosyringone (4-hydroxy-3,5-dimethoxy-acetophenone), vanillic acid and syringic acid, occurring in 0.04-0.12 g per 100 g straw concentrations. High lignin removal from the solid fraction (62%) did not provide a corresponding increase in the phenol monomer content but was correlated to high carboxylic acid concentrations. The degradation products in the hemicellulose fractions co-varied with the pre-treatment conditions in the principal component analysis according to their chemical structure, e.g. diacids (oxalic and succinic acids), furan aldehydes, phenol aldehydes, phenol ketones and phenol acids. Aromatic aldehyde formation was correlated to severe conditions with high temperatures and low pH. Apart from CO2 and water, carboxylic acids were the main degradation products from hemicellulose and lignin.     

Mapping of AFLP markers linked to seed coat colour loci in<Emphasis Type="Italic"> Brassica juncea</Emphasis> (L.) Czern

Association mapping of the seed-coat colour with amplified fragment length polymorphism (AFLP) markers was carried out in 39 Brassica juncea lines. The lines had genetically diverse parentages and varied for seed-coat colour and other morphological characters. Eleven AFLP primer combinations were used to screen the 39 B. juncea lines, and a total of 335 polymorphic bands were detected. The bands were analysed for association with seed-coat colour using multiple regression analysis. This analysis revealed 15 markers associated with seed-coat colour, obtained with eight AFLP primer combinations. The marker E-ACA/M-CTG₃₅₀ explained 69% of the variation in seed-coat colour. This marker along with markers E-AAC/M-CTC₂₃₅ and E-AAC/M-CTA₂₅₀ explained 89% of the total variation. The 15 associated markers were validated for linkage with the seed-coat colour loci using a recombinant inbred line (RIL) mapping population. Bands were amplified with the eight AFLP primer combinations in 54 RIL progenies. Of the 15 associated markers, 11 mapped on two linkage groups. Eight markers were placed on linkage group 1 at a marker density of 6.0 cM, while the remaining three were mapped on linkage group 2 at a marker density of 3.6 cM. Marker E-ACA/M-CTG₃₅₀ co-segregated with Gene1 controlling seed-coat colour; it was specific for yellow seed-coat colour and mapped to linkage group 1. Marker E-AAC/M-CTC₂₃₅ (AFLP8), which had been studied previously, was present on linkage group 2; it was specific for brown seed-coat colour. Since AFLP markers are not adapted for large-scale applications in plant breeding, it is important to convert these to sequence-characterised amplified region (SCAR) markers. Marker E-AAC/M-CTC₂₃₅ (AFLP8) had been previously converted into a SCAR. Work is in progress to convert the second of the linked markers, E-ACA/M-CTG₃₅₀, to a SCAR. The two linked AFLP markers converted to SCARs will be useful for developing yellow-seeded B. juncea lines by means of marker-assisted selection.Communicated by H.F. Linskens     

Isoelectric focusing electrophoresis of lignin.

Isoelectric focusing is introduced as a technique for the analysis of macromolecular lignin. The analysis is performed in a pH gradient from 3.5 to 10. Separated lignin fragments are visualized under uv light or by silver staining. The method can be used to distinguish between differently processed lignin preparations and to identify their components. Even the slight modification resulting from attack by ligninolytic enzymes could be detected.