Selective Delignification Research Articles

Roles of Diverse Fungi in Larch Needle-Litter Decomposition

Functional biodiversity of fungi in larch (Larix leptolepis) forests needle-litter decomposition was examined by a pure-culture test. Weight loss of larch-needle litter, utilization pattern of lignocellulose and chemical composition of remaining litter were investigated and compared for 31 isolates in 27 species of basidiomycetes and ascomycetes. Weight loss (% original weight) of litter ranged from −2.0% to 14.2%. Mean weight loss of litter caused by the basidiomycetes was not significantly different from that caused by the ascomycetes. Basidiomycetes caused loss of lignin and carbohydrates in variable proportions, while ascomycetes exclusively attacked carbohydrates without delignification. The content of lignin and nitrogen in remaining litter was not significantly correlated when both basidiomycetes and ascomycetes were included. However, the correlation coefficient was significant when the relationship was examined separately for basidiomycetes, indicating that the degree of selective delignification determined the final nitrogen content in litter. Possible effects of fungal colonization on needle-litter decomposition in larch forests are discussed.

Roles of diverse fungi in larch needle-litter decomposition

Functional biodiversity of fungi in larch (Larix leptolepis) forests needle-litter decomposition was examined by a pure-culture test. Weight loss of larch-needle litter, utilization pattern of lignocellulose and chemical composition of remaining litter were investigated and compared for 31 isolates in 27 species of basidiomycetes and ascomycetes. Weight loss (% original weight) of litter ranged from −2.0% to 14.2%. Mean weight loss of litter caused by the basidiomycetes was not significantly different from that caused by the ascomycetes. Basidiomycetes caused loss of lignin and carbohydrates in variable proportions, while ascomycetes exclusively attacked carbohydrates without delignification. The content of lignin and nitrogen in remaining litter was not significantly correlated when both basidiomycetes and ascomycetes were included. However, the correlation coefficient was significant when the relationship was examined separately for basidiomycetes, indicating that the degree of selective delignification determined the final nitrogen content in litter. Possible effects of fungal colonization on needle-litter decomposition in larch forests are discussed.

Effects of prior decomposition of beech leaf litter by phyllosphere fungi on substrate utilization by fungal decomposers

Changes in litter quality resulting from pretreatment of leaf litter by phyllosphere fungi may affect its subsequent decomposition by succeeding fungi. The purpose of this study is to clarify the effect of prior decomposition of leaf litter by two phyllosphere fungi of beech, Xylaria sp. and Ascochyta sp., on substrate utilization of 12 fungal species in the Basidiomycota, the Ascomycota, and the Zygomycota, in a laboratory experiment. Mycena sp. caused significantly higher weight loss in litter previously partly decomposed by Xylaria sp. than in control litter without fungal inoculation and litter previously partly decomposed by Ascochyta sp., whereas prior decomposition retarded litter decomposition or had no significant effect on 11 other species. Prior decomposition by phyllosphere fungi affected the substrate utilization patterns of two Mycena species in the Basidiomycota, shifting from simultaneous removal of lignin and carbohydrates to selective delignification.

Alkaline pulping with additives of kenaf from Sudan

Soda-anthraquinone (soda-AQ), alkaline sulfite-anthraquinone (AS-AQ) and ASAM (alkaline sulfite-anthraquinone-methanol) cooking of kenaf core, bark and whole stalk from Sudan was carried out under different conditions and pulps with good to very good yields and mechanical properties were obtained. The AS-AQ process was particularly well suited for kenaf whole stalk and bark with yield, viscosity, brightness and strength properties superior to those of soda and soda-AQ pulping. ASAM pulping provided a further selective delignification with higher yield, lower κ number and superior viscosity and brightness at high pulp strength level. Blending 90 and 70% kenaf core pulp with pulp from kenaf bark improved all the strength and optical properties, with the 70% blends better. The application of a totally chlorine free (TCF) bleaching sequence of OQ 1(PO)Q 2P on whole kenaf stalk soda-AQ and ASAM pulps gave pulp very high brightness and strength properties.

Equilibrating metal-oxide cluster ensembles for oxidation reactions using oxygen in water.

Although many enzymes can readily and selectively use oxygen in water-the most familiar and attractive of all oxidants and solvents, respectively-the design of synthetic catalysts for selective water-based oxidation processes utilizing molecular oxygen remains a daunting task. Particularly problematic is the fact that oxidation of substrates by O2 involves radical chemistry, which is intrinsically non-selective and difficult to control. In addition, metallo-organic catalysts are inherently susceptible to degradation by oxygen-based radicals, while their transition-metal-ion active sites often react with water to give insoluble, and thus inactive, oxides or hydroxides. Furthermore, pH control is often required to avoid acid or base degradation of organic substrates or products. Unlike metallo-organic catalysts, polyoxometalate anions are oxidatively stable and are reversible oxidants for use with O2 (refs 8,9,10). Here we show how thermodynamically controlled self-assembly of an equilibrated ensemble of polyoxometalates, with the heteropolytungstate anion [AIVVW11O40]6- as its main component, imparts both stability in water and internal pH-management. Designed to operate at near-neutral pH, this system facilitates a two-step O2-based process for the selective delignification of wood (lignocellulose) fibres. By directly monitoring the central Al atom, we show that equilibration reactions typical of polyoxometalate anions keep the pH of the system near 7 during both process steps.

Nitrogen-removal with protease as a method to improve the selective delignification of hemp stemwood by the white-rot fungus Bjerkandera sp. strain BOS55

Certain white-rot fungi cause selective removal of lignin from woody substrates. Selective delignification can potentially be applied to biopulping and upgrading animal feeds. Nitrogen nutrient limitation is known to enhance the selectivity of lignin degradation. The relatively high N-content of annual fiber crops is an important drawback for utilizing white-rot fungi for their selective delignification. In this study, removal of N from hemp stemwood with protease was explored as a means of improving the selectivity of lignin degradation by the white-rot fungus Bjerkandera sp. strain BOS55. Various protease treatments followed by hot-water extraction were found to be suitable in lowering the N-content of hemp stemwood by up to 70%. The removal was significantly higher than with hot-water extraction alone, which caused a 39% N-removal. The selectivity of lignin degradation was compared in protease-treated, hot-water treated, untreated and ammonium-spiked hemp stemwood, providing N levels that were, respectively, 0.32-, 0.61-, 1.0- and 5.0-fold relative to the natural N-content in the substrate. Removal of N by hot-water extraction alone or in combination with protease greatly protected the holocellulose fraction from excessive decay during 10 weeks of solid state fermentation. However, the selectivity of lignin decay was only greatly enhanced (three-fold) by the protease treatment, due mostly to a highly improved lignin degradation at the lowest N-level.

Infrared spectroscopy analysis of hemp (Cannabis sativa) after selective delignification by Bjerkandera sp. at different nitrogen levels.

Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor changes in C/N-modified lignocellulosic substrates from Cannabis sativa L. in a 7-week solid-state fermentation with the white-rot fungus Bjerkandera sp. strain BOS55. The microbial transformation of hemp was considered as a pretreatment to pulping processes in paper industries. Special emphasis was paid on the N-content of the substrate, which was modified by: (i) external ammonium inputs, (ii) water extraction, and (iii) protease treatment.Selective delignification in the N-limited media was observed. The most diagnostic FT-IR spectral bands in relation to changes in the lignocellulosic substrate were those corresponding to alkyl structures (2920, 1460 cm(-1)), carboxyl groups (1720 cm(-1)), amides (1650, 1540 cm(-1)) and carbohydrate (mainly 1030 cm(-1)). Simple and multiple regression functions revealed the potential of FT-IR in accurately reflecting substrate composition features previously determined by wet chemical methods. Correspondence analysis suggests C/N-dependent degradation patterns, and discriminant analysis confirmed that the differences between N-limited, N-enriched and the original substrate were significant (P < 0.05) in terms of the intensities of five FT-IR diagnostic bands (1030, 1130, 1270, 1540 and 1650 cm(-1)).The results suggest that, in the system studied, the FT-IR spectroscopy is a reliable alternative to wet chemical analyses in the routine monitoring of the success of the biologic process since it reflects both qualitative and quantitative changes and it is very sensitive to lignin alteration and to carbohydrate and protein concentration.

Anatomical Study of the Decay Caused by the White-Rot Fungus Trametes Trogii (Aphyllophorales) in Wood of Salix and Populus

Different stages of decay caused in vitro by Trametes trogii in Salix sp. and Populus sp. wood are described. Anatomical features are reported in three stages of this process. Decay progressed in a different pattern in both species studied. In Populus sp. T. trogii caused a combination of selective delignification and simultaneous decay within the same substrate. In advanced stages wood blocks exhibited large empty holes and a spongy structure. In Salix sp. a simultaneous white-rot decay took place. Only vessels remained and the residual white-rotted wood developed a stringy appearance.

Comparison of wood decay among diverse lignicolous fungi

In decay tests with 98 isolates (78 species) of lignicolous fungi followed by chemical and anatomical analyses, the validity of the generally accepted, major decay types (white, brown, and soft rot) was confirmed, and no new major types proposed. We could distinguish soft rot from other decay types based on anatomical and chemical criteria, without reliance on cavities or recourse to taxonomy of causal agents. Chemically, soft rot of birch could be distinguished from white rot by lower Klason lignin loss, and from brown rot by much lower alkali solubility. Anatomically, erosion of birch fiber walls in soft rot was distinguished from that in white rot by the angular erosion channels, V-shaped notches and diamond-shaped, eroded pit apertures that predominated in the former and their rounded forms in the latter.Substantial decay was caused by fungi representing eight orders in addition to the Aphyllophorales. Members of the Exidiaceae generally caused low weight losses and anatomical and chemical patterns of degradation characteristic of white rot. Isolates of Auricularia auricula-judae also caused a white rot, with high weight losses and unusual, branching microcavities that were oriented longitudinally in the S2 cell-wall layer. Ten species of the Dacrymycetales caused a brown rot like that caused by some Aphyllophorales; most caused high weight losses.Among white-rot fungi on birch, a relationship was observed between strongly selective delignification and strongly selective utilization of mannose. Among brown-rot fungi on birch, the top two polyose sugars (not including glucose) in order of selectivity were galactose>mannose; among soft-rotters they were arabinose>xylose. On pine, distinctions were not so clear, but some differing trends were evident.Previously unreported selective delignifiers were found in the Auriculariales, Agaricales, and in two orders of gasteromycetes. Selective delignification was most pronounced at low weight losses. Certain decay features similar to those in the Ascomycota were found in the Auriculariales, consistent with hypotheses that place that order near the phylogenetic root of Basidiomycota. A sequence of origins of decay types is proposed.

The Effects of Hydrogen Peroxide and Anthraquinone on Soda Ash Pulping of Wheat Straw

A comparative study was undertaken to evaluate the effects of H 2 O 2 and AQ on wheat straw pulping. The alkaline pulping liquor was a mixture of Na 2 CO 3 and NaOH at the ratio of 7 to 3. The addition of 0.1% AQ to the cooking liquor reduced the alkali consumption by about 34% and decreased the Kappa number by about 20 units. By contrast, the addition of 3% H 2 O 2 increased the alkali consumption by about 25% and did not increase the degree of delignification. The addition of AQ resulted in more selective delignification while the addition of H 2 O 2 is less selective. Na 2 CO 3 /NaOH/AQ process (AP-AQ) is found suitable for pulping of wheat straw, producing a high yield bleachable grade pulp.

Effects of Incubation Time and Temperature on In Vitro Selective Delignification of Silver Leaf Oak by Ganoderma colossum

The effects of incubation time and temperature on the ability of isolates of the chlamydosporic and thermophilic fungus Ganoderma colossum (Fr.) C. F. Baker to cause selective delignification of Quercus hypoleucoides A. Camus were evaluated by standard in vitro agar block tests. Chemical and scanning electron microscopy studies of decayed wood were used to determine the extent of selective delignification or simultaneous decay caused by each fungal isolate. At 35 deg C, the percent weight loss increased from 6.1% after 4 weeks to a maximum of 32.5 to 33.0% after 16 and 20 weeks of incubation. The average percent Klason lignin-chlorite holocellulose ratios (PKL/CHC) decreased from 0.35 in the control wood block to 0.22 in wood blocks incubated for 12 weeks; this indicated selective delignification. The average PKL/CHC increased for the 16- and 20-week incubation periods, indicating greater removal of polysaccharides during longer incubation periods. In temperature studies, the percent weight loss after 12 weeks was 26 to 27% between 30 and 40 deg C and less than 16% for the 25 and 45 deg C treatments. The average PKL/CHC ranged from 0.18 to 0.16 between 35 and 40 deg C, whereas they were 0.23 and 0.31 for the 25 and 45 deg C treatments, respectively. Scanning electron microscopy confirmed an optimum temperature range near 35 to 40 deg C and incubation times of 8 to 12 weeks for selective delignification. Under these conditions, ray parenchyma, fiber tracheids, and vessels were devoid of middle lamella; pit regions of cells were visible with significantly enlarged apertures; and individual cells were separated and clearly delimited. Extensive delignification of wood occurred throughout the wood blocks evaluated. Incubation times longer than 12 weeks resulted in greater degradation of wood cell walls and thus in greater removal of the polysaccharide component of the wood. For incubation times of 4 weeks or a temperature of 25 deg C, limited to no degradation of cells was observed. At 45 deg C, walls of fiber tracheids were eroded and ray parenchymal cells were extensively degraded, indicating that simultaneous degradation of cell walls occurred. Thus, the incubation temperature influenced the type of decay by G. colossum observed on oak wood blocks: extensive selective delignification at 35 to 40 deg C after more than 8 weeks of incubation or simultaneous decay at 45 deg C with 14% weight loss after 12 weeks of incubation. Isolates of G. colossum may prove useful in studies on mechanisms of delignification and biotechnological applications (e.g., biopulping) of lignin-degrading fungi.

A Simple Method That Uses Differential Staining and Light Microscopy To Assess the Selectivity of Wood Delignification by White Rot Fungi

CRYOSTAT MICROTOME SECTIONS OF BIRCH WOOD DEGRADED BY WHITE ROT FUNGI WERE EXAMINED BY LIGHT MICROSCOPY AFTER TREATMENT WITH TWO STAINS: astra-blue, which stains cellulose blue only in the absence of lignin, and safranin, which stains lignin regardless of whether cellulose is present. The method provided a simple and reliable screening procedure that distinguishes between fungi that cause decay by selectively removing lignin and those that degrade both cellulose and lignin simultaneously. Moreover, morphological characteristics specific to selective delignification were revealed.

Microstructural and Histochemical Changes in Acer platanoides Rhytidome Caused by Dendrothele acerina (Aphyllophorales) and Mycena meliigena (Agaricales)

Rhytidome blocks from Acer platanoides were incubated in vitro with Dendrothele acerina or Mycena meliigena mycelia in order to describe resultant microstructural and histochemical changes using paraffin sections and brightfield microscopy. Rhytidome consists of alternating layers of periderm and nonfunctional secondary phloem. All cells in the rhytidome, except suberized phellem cells, are lignified. Both fungi penetrated blocks, and advanced 1–2 millimeters in 20 weeks. Both species penetrated suberized phellem layers and eroded phellem cell walls from the lumen outward. In nonfunctional phloem, hyphae moved between lumina of the sieve tube elements, companion cells, and parenchyma through enlarged sieve pores, pits, and bore holes. These walls were progressively thinned from the lumen outward. Fiber and sclereid walls were penetrated through pits and bore holes, but the hyphae remained in the middle of the secondary wall, forming crescent-shaped cavities that gradually enlarged to leave a honeycomb pattern of compound middle lamellae. Both fungi displayed selective delignification and nonselective cell wall degradation patterns observed in some white rot fungi. We suggest that D. acerina and M. meliigena should be classified ecologically as saprotrophic inhabitants of the rhytidome of living trees.

Optimisation of solid substrate fermentation of wheat straw into animal feed by Pleurotus ostreatus: a pilot effort

The solid substrate fermentation (SSF) of wheat straw with the selected white-rot fungus, Pleurotus ostreatus, was optimised for its cultural and nutritional conditions and substrate pretreatment based on selective delignification (more lignin than holocellulose degradation), crude protein content and in vitro dry matter digestibility (IVDMD). The differences between treatments were found to be statistically significant ( P ⩽ 0.01). The fungus behaved optimally under the following set of fermentation conditions: initial pH, 5.5; initial moisture, 55%; temperature, 22 °C; period, 21 days; form of inoculum-grain culture (spawn); substrate turning frequency, once at mid-incubation; urea (nitrogen source) level, 1% (sterile) or 2% (unsterile); single superphosphate (phosphorus + sulphur source), 0.3%; no addition of free carbohydrates (as whey or molasses). A maximum (10.4%) increase in IVDMD accompanied by a 2.7% (g per 100 g dry matter of original straw) degradation of lignin was attained in the optimised SSF under the above conditions. The digestibility could, however, be further improved by using a two-stage process involving pretreatment (physical/chemical/biological) before fermentation. For practical farm applications, urea treatment and ensiling to be the most feasible pretreatments. The laboratory scale (40-g lots) optimised SSF was also scaled-up to 4 kg (cooked and uncooked) and 50 kg (uncooked) fermentations.

Phellinus ralunensis (aphyllophorales: Hymenochaetaceae), a new white pocket rot species from Chile

A new fungal species, Phellinus ralunensis , from Ralun, Chile is described based on basidiome, cultural, and wood decay characteristics. Distinctive characteristics include clusters of trichocysts on the pore surface of basidiomes and circinate vesicles in culture. Optimum temperature range for growth is 20–25 °C, with no growth ≥ 30°. The fungus causes a white pocket rot resulting in localized areas of selective delignification in wood of Eucryphia cordifolia .

Optimization of cultivation and nutrition conditions and substrate pretreatment for solid-substrate fermentation of wheat straw byCoriolus versicolor

Bioconversion of wheat straw by solid-substrate fermentation (SSF) with Coriolus versicolor was optimized by varying its physiological parameters. Selective delignification (more lignin than holocellulose degradation) and increases in crude protein (CP) content and in vitro dry matter digestibility (IVDMD) were taken as the criteria to select optimum levels of these parameters. The fungus behaved optimally under the following set of cultural and nutritional conditions: pH 5.5, moisture level 55%, temperature 30 degrees C, duration of fermentation 21 d, form of inoculum--grain culture, turning frequency--once at mid-incubation, urea (nitrogen source) 1.5% (sterile) or 3.0% (nonsterile), single superphosphate (phosphorus + sulfur source) 1.0%, no addition of free polysaccharides (as whey or molasses). A maximum of 17.5% increase in IVDMD involving 4.3% degradation of lignin, was attained in the optimized SSF under laboratory conditions. The digestibility improvement could be further increased by using a substrate pretreatment (physical/chemical/biological) in the following order of preference: NaOH treatment, urea or urine treatment, ensiling, steaming, grinding. For practical farm applications, urea treatment and ensiling appeared most feasible. The laboratory optimized process was also scaled up to 4 kg (sterile and unsterile) and 50 kg (unsterile) fermentations.

Decay of date palm wood by white-rot and brown-rot fungi

Wood from trunks of Canary Island date palm (Phoenix canariensis) was decayed for 12 weeks with white-rot fungi (Ganoderma colossum, G. zonatum, Phanerochaete chrysosporium, Scytinostroma galactinum, or Trametes versicolor) or brown-rot fungi (Wolfiporia cocos, Gloeophyllum trabeum, or Fomitopsis pinicola). Using the vermiculite-block assay, white-rot fungi caused significantly more weight loss (63%) than brown-rot fungi (32%). Of the white-rot fungi, G. colossum caused the greatest weight loss (81%), while S. galactinum caused the least (36%). In contrast, weight loss caused by the brown-rot fungi was similar. Chemical analyses indicated that both white-rot and brown-rot fungi caused losses of starch, holocellulose, and lignin. White-rot fungi, however, removed greater amounts of lignin than the brown-rot fungi with three species, S. galactinum, P. chrysosporium, and G. zonatum, causing selective delignification. Scanning and transmission electron microscopy showed that phloem and parenchyma cells were more susceptible to decay than xylem and fiber cells. Starch grains were degraded by all fungi and were nearly removed in wood decayed by G. colossum. In wood decayed by white-rot fungi, cell walls were eroded and middle lamellae were degraded. Selective delignification was observed in fibers adjacent to vascular tissue in wood decayed by the three white-rot fungi. In wood decayed by brown-rot fungi, walls of ground parenchyma and vascular bundle cells were swollen and fragmented when physically disrupted. In wood decayed by F. pinicola, some cell walls were nearly disintegrated. Key words: selective delignification, simultaneous decay, ultrastructure.

Characterization of Palo Podrido, a Natural Process of Delignification in Wood

Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of Eucryphia cordifolia and Nothofagus dombeyi, which have the lowest lignin content and whose lignins have the largest amount of beta-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A Ganoderma species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the beta-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified N. dombeyi wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan.

Optimization of solid-state fermentation for selective delignification of aspen wood with Phlebia tremellosa

The white-rot fungus Phlebia tremellosa can delignify aspen wood and increase the accessibility of its polysaccharides to enzymatic hydrolysis, under solid-state fermentation conditions. Fermentations at the 50 g scale were conducted to provide information for the design of larger fermentations. Agitation was not required. Forced aeration was needed for delignification of wood layers more than a few millimeters thick, but air circulation between the particles was not blocked by mycelium. Shavings were the best particles size. Sterilization of the wood was essential, but preadaptation of the inoculum to growth in wood was not. Inoculum levels as low as 2% were adequate.

Delignification of Wood Chips and Pulps by Using Natural and Synthetic Porphyrins: Models of Fungal Decay

Kraft pulps, prepared from softwoods, and small chips of birch wood were treated with heme and tert-butyl hydroperoxide in aqueous solutions at reflux temperature. Analyses of treated pulps showed decreases in kappa number (a measure of lignin content) from about 36 to less than 2, with concomitant increases in brightness (80% increase in the better samples). Analyses of treated wood chips revealed selective delignification and removal of hemicelluloses. After 48 h of treatment, lignin losses from the wood chips approached 40%, and xylose/mannose (hemicellulose) losses approached 70%, while glucose (cellulose) losses were less than 10%. Examination of delignified chips by transmission electron microscopy showed that the removal of lignin occurred in a manner virtually indistinguishable from that seen after decay by white rot fungi. Various metalloporphyrins, which act as biomimetic catalysts, were compared to horseradish peroxidase and fungal manganese peroxidase in their abilities to oxidize syringaldazine in an organic solvent, dioxane. The metalloporphyrins and peroxidases behaved similarly, and it appeared that the activities of the peroxidases resulted from the extraction of heme into the organic phase, rather than from the activities of the enzymes themselves. We concluded that heme-tert-butyl hydroperoxide systems in the absence of a protein carrier mimic the decay of lignified tissues by white rot fungi.