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) withCoriolus versicolor was optimized by varying its physiological parameters. Selective delignification (more lignin than holocellulose degradation) and increases in crude protein (CP) content andin 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 °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 preteatment (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.     

The effectiveness of biological treatment of wheat straw by white-rot fungi

Out of 13 species ofBasidiomycetes growing on wheat straw, 9 species enhanced thein vitro dry matter digestibility of the substrate. The detergent fiber content (acid and neutral) of the substrate was significantly reduced by most of the fungi tested. Hemicellulose showed the largest proportionate loss, whereas lignin the smallest one.     

Biodegradation of wheat straw lignocarbohydrate complexes (LCC)

Summary In laboratory and semi-industrial scale experiments the influence of the substrate water content, temperature, and incubation time on the progress of solid state fermentation of straw colonized by white rot fungi was investigated. The parameters used to evaluate the fermentation process were degradation of total organic matter and lignin, in vitro digestibility, the content of water soluble substances in the substrate and the pH.The degradation of total organic matter was species specific. Only Trametes hirsuta enhanced the degradation at elevated temperature (30 °C). With Abortiporus biennis, Ganoderma applanatum, and Pleurotus serotinus, elevated temperature had and adverse effect. Prolonged incubation only improved degradation of straw by the relatively slowgrowing fungi Ganoderma applanatum, Lenzites betulina, and Pleurotus sajor caju.Elevated temperature and prolonged incubation shifted the relative degradation rates in favour of total organic matter degradation. With Ganoderma applanatum, Pleurotus ostreatus, and Pleurotus serotinus lignin degradation, even on an absolute scale, was less at 30 °C than at 22 °C.In general, the in vitro digestibility also decreased, when the incubation time and temperature were raised. With Ganoderma applanatum the in vitro digestibility dropped below the value of the sterile straw control.Solid state fermentation of straw was at an optimum at a medium water content of 75 ml/25 g of substrate. However, most of the fungi tested could digest straw over a wide range of water content. At higher water contents (125–150 ml/25 g of substrate) an increased production of aerial mycelium was observed.In semi-industrial batch experiments (40 kg) with Abortiporus biennis the in vitro digestibility dropped below the reference value for sterile straw during the first 19 days of incubation. Later, the in vitro digestibility again rose and reached its optimum after about 60 days. The in vitro digestibility in the semi-industrial experiments was always lower than in the laboratory experiments (+9% and +25%, respectively).In long term experiments (2.5 kg batches, 8 months of incubation) very different values for the in vitro digestibility were found, and these depended on the fungus used (Abortiporus biennis, +16%; Pleurotus ostreatus, +4%; and Ganoderma applanatum, –27%).     

Influence of oxygen and carbon dioxide on lignin degradation in solid state fermentation of wheat straw withStropharia rugosoannulata

Summary In solid state fermentation,Stropharia rugosoannulata degrades lignin of wheat straw slightly better in the presence of oxygen than that in air. The sub-atmospheric partial pressure of oxygen (0.05 atm.) inhibits lignin and organic matter degradation. the increasing partial pressure of carbon dioxide (0.1–0.3 atm.) along with 0.2 atm. of oxygen does not have any effect on lignin degradation, but slightly decreases organic matter loss and increases thein vitro digestibility of fermented wheat straw, thereby making the process more efficient.     

Solid state fermentation of lignocellulose containing plant residues withSporotrichum pulverulentum Nov. andDichomitus squalens (Karst.) Reid.

Summary The fermentation profiles ofSporotrichum pulverulentum andDichomitus squalens showed distinct differences.D. squalens digested the substrate more slowly thanSporotrichum pulverulentum. The relative degradation rates of total organic matter and lignin also differed considerably.Whereas withS. pulverulentum the ratio was about the same throughout the whole observation period, withDichomitus squalens it altered in favour of lignin degradation.WithSporotrichum pulverulentum an optimum digestion in vitro of 40%–50% was achieved after 20 days of incubation. WithDichomitus squalens the best value (about 60%) was reached after 30 days of incubation. Increasing incubation temperatures enhanced the degradation of the substrate.As found with wheat straw, all other substrates tested (straw of rape and barley, glumes of rice) were degraded more slowly byDichomitus squalens than bySporotrichum pulverulentum. The degradation rates for oak, spruce and beech sawdust were very low compared to those for straw.Small amounts of ammonium nitrate stimulated the degradation of straw byS. pulverulentum whereas higher concentrations had an inhibitory effect. The optimum water content of the substrate, measured by decomposition of total organic matter and lignin and by in vitro digestibility, was between 50 and 100 ml of water/25 g substrate. Higher and lower water contents had an unfavourable effect.Varying the pore size of the substrate by using milled straw of defined particle size had no influence on the 6 parameters tested under the given experimental conditions.The best method to supress potential competitors was to heat the substrate to 90°C for 24 h.     

Substrate-dependent degradation patterns in the decay of wheat straw and beech wood by ligninolytic fungi

Summary The ability of 45 fungal strains to degrade wheat straw and beech wood was studied. Degradation patterns were defined in terms of chemical evolution of substrates and changes in lignin and polysaccharides. Trametes versicolor produced an important degradation of lignin and increased substrate digestibility, but it caused high weight losses and gave rise to similar decay patterns on both substrates. A preferential degradation of lignin was produced during straw transformation by Pleurotus eryngii. The increase of soluble lignin and decreases of lignin content and H/C ratio defined the degradation tendency after principal component analysis. The cation exchange capacity and water and alkali solubility presented the highest loading factors for the characterization of fungal transformation of beech wood. Offprint requests to: A. T. Martínez     

The influence of ammonium nitrate supplementation on degradation and in vitro digestibility of straw colonized by higher fungi

Summary The digestion of straw by nine higher fungi and the influence of ammonium nitrate supplementation was investigated using loss of organic matter, degradation of lignin and in vitro digestibility as indices of digestion.The decomposition rates of organic matter were influenced differently by ammonium nitrate supplementation. The rates of decomposition by Pleurotus salmoneo stramineus and Pleurotus eryngii decreased, while those of Lentinus edodes and Pleurotus sp. Florida increased at low NH₄NO₃ concentrations, but decreased at higher concentrations. The decomposition rates of Agrocybe aegerita, Stropharia rugosoannulata, Kuehneromyces mutabilis, and Ganoderma applanatum were generally higher than those of the control at all NH₄NO₃ levels.The ability to degrade lignin varied greatly. Flammulina velutipes did not attack lignin at all while Agrocybe aegerita did only slightly. The highest lignin degradation rate was found with Pleurotus sp. Florida. The lignin decomposition rates of Lentinus edodes, Pleurotus eryngii and Pleurotus salmoneo stramineus were depressed at all NH₄NO₃ levels. No correlation between lignin degradation and nitrogen supplementation was found with Ganoderma applanatum and Pleurotus sp. Florida. Only Agrocybe aegerita could be stimulated with NH₄NO₃.The in vitro digestibility of the substrate with and without nitrogen supplementation was decreased with Agrocybe aegerita, Flammulina velutipes, and Ganoderma applanatum. All the other fungi enhanced the in vitro digestibility when no NH₄NO₃ was added to the substrate. At all NH₄NO₃ levels, the in vitro digestibility was lower than that of the unsupplemented control. At 1.25% NH₄NO₃ it fell below the fungus free control value.The results are explained in terms of altered relative decomposition rates for organic matter and lignin after nitrogen supplementation.     

The effects of co-fungal cultures and supplementation with carbohydrate adjuncts on lignin biodegradation and substrate digestibility

The ability of three fungal strains (Pleurotus sajor-caju, Phanerochaete chrysosporium, Trametes versicolor) to decrease the lignin content and to enhance in vitro rumen digestibility of lignified spruce sawdust was assessed. In monoculture solid substrate fermentation (SSF) studies, a considerable length of time (6 weeks) elapsed before 4 to 14% lignin was degraded. In contrast, paired or multiple cultures of these fungi caused an 8 to 16% loss of native lignin within three weeks of incubation. There were also synergistic effects on total polysaccharide/hemicellulose degraded by mixed cultures. A similar observation was made for in vitro digestibility of fungal fermented samples: Total solubles (carbohydrate products) which accumulated in cultures were significantly higher in mixed cultures than in respective monocultures. In contrast, mixtures of cell free enzyme extracts of these fungi did not cause any marked reduction in lignin or cellulose content. Supplementation of wood sawdust with carbohydrate adjuncts prior to fungal treatment also led to substantial reduction in lignin content and increased substrate digestibility.F.O. Asiegbu is with the Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, P.O. Box 7026, S-750 07 Uppsala, Sweden; A. Paterson and J.E. Smith are with the Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, G1 1XW, UK.     

Bioconversion of sugar cane crop residues with white-rot fungiPleurotus sp.

Four mushroom strains ofPleurotus spp. were cultivated on sugar cane crop residues for 30 days at 26°C. Biochemical changes affected the substrate as a result of fungal growth, in terms of nitrogen, lignin, cellulose and hemicellulose contents. All strains showed a strong ligninolytic activity together with variable cellulolytic and xylanolytic action.Pleurotus sajor-caju attacked lignin and cellulose at the same rate, showing a degradation of 47% and 55%, respectively. A better balance was shown by theP. ostreatus-P. pulmonarius hybrid, which exhibited the poorest cellulolytic action (39%) and the highest ligninolytic activity (67%). The average composition of mushroom fruit bodies, in terms of nitrogen, carbohydrates, fats and amino acid profiles, was determined. Crude protein and total carbohydrate varied from 23% to 33% and 36% to 68% of dry matter, respectively. Fat ranged from 3.3% to 4.7% and amino acid content from 12.2% to 22.2%. Slight evidence for a nitrogen fixing capability was encountered in the substrate to fruit body balance.     

Utilization of lignocellulosic waste by the edible mushroom,Pleurotus

Lignocellulosic waste represents huge amounts of unutilized renewable resource. The use of the polysaccharides in the lignocellulosic complex is limited due to their high lignin content. White rot fungi are capable of selectively degrading lignin, thereby upgrading it. The focus of this article is on the potential utilization of edible mushrooms of the genus Pleurotus, via solid state fermentation, using cotton plant stalks as a substrate. This material poses agrotechnical problems since the stalks have a fibrous structure similar to that of hardwood. Potential uses for this material are as a fuel in rural areas, a substrate for mushrooms, an animal feed and substrate for paper making. In this study, degradation of cotton stalks by Pleurotus is described using chemical analyses and scanning electron microscopy. During four weeks of solid state fermentation, lignin content significantly decreased and in vitro digestibility was increased. The fermentation product was consumed by ruminants at a level of up to 40% of their diet.     

Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

Bioconversion of sugarcane bagasse with white rot fungi

Summary Four cultures of white rot fungi were screened for their ability to degrade lignin and carbohydrates of sugarcane bagasse and their effect on changes inin vitro digestibility.Polyporus hirsutus534 degraded maximum lignin and carbohydrates accompanied with the highest increase in digestibility, but increase in nutrient availability was maximum withPleurotus sajorcaju (Z-6) due to lower dry matter loss during the process of fungal treatment. All the fungi tested exceptPolyporus caperatus Berk. degraded lignin more selectively than the other components of sugarcane bagasse.     

Effect of different supplements on bioprocessing of wheat straw by Phlebia brevispora: changes in its chemical composition, in vitro digestibility and nutritional properties

Bioprocessing of wheat straw was carried out by Phlebia brevispora under solid state conditions. Effect of different supplements on lignocellulolytic enzymes production, degradation of straw cell wall fibers and its resultant effect on nutritional quality of wheat straw were studied. Ammonium chloride and malt extract were more effective in terms of ligninolysis and enhanced in vitro digestibility. The concentration of the selected supplements and the moisture content was worked out using response surface methodology in order to minimize the loss in total organic matter so as to selectively degrade lignin. The experiment was scaled up to batches of 200 g under optimized conditions and the degraded substrate was analyzed for its biochemical properties. P. brevispora degraded 290 g/kg of lignin and enhanced the in vitro digestibility from 150 to 268 g/kg (78%). Crude protein, amino acids, total phenolic contents and antioxidant properties were significantly higher in degraded straw.     

Degradation of alkali lignin by two ascomycetes and free radical scavenging activity of the products

Biodegradation and bioconversion of extracted alkali lignin was performed under varying concentrations of carbon and nitrogen sources, by two potential Ascomycetes ligninolytic fungus isolated from soil. Fungus, F10 was identified as Aspergillus flavus, while APF4 as Emericella nidulans based upon closed similarity with their morphology and high homology in 18S rRNA gene sequences. The alkali lignin degradation was checked in term of disappearance of lignin content and colority. Selected fungus, degraded 19–41.6% of alkali lignin (0.25%, w/v) within 21 days of incubation and reduced the colority up to 14.4–21%. The activity of ligninolytic enzymes was periodically checked. During alkali lignin degradation manganese peroxidase (13.31?U/ml), lignin peroxidase (13.73?U/ml) and laccase (0.05?U/ml) activities were observed (at highest level). The alkali lignin degradation products and functional group changes in degraded lignin were analysed through gas chromatography-mass spectroscopy (GC-MS) and solid state ¹³C-NMR spectroscopy, respectively. The functional group modifications in alkali lignin moiety, alter its biochemical property, thus fungal mediated modified alkali lignin was further tested for reactive free radical scavenging potential with respect to hydroxyl, nitric oxide and superoxide radicals. Results demonstrate that the alkali lignin undergo degradation in studied nutritional conditions (high-carbon low nitrogen) and consequently increase its free radical scavenging activity up to 1–18%.     

Growth of higher fungi on wheat straw and their impact on the digestibility of the substrate

Summary The influence of the growth of three higher fungi on the composition of wheat straw was investigated. Pleurotus pulmonarius, P. sajor-caju and Lentinus edodes grew very well on lignocellulosic substrates, breaking down a considerable amount of lignin. The initial lignin concentration of straw was halved after 12 weeks of fungal growth, doubling the enzymic digestibility. Together with lignin, the higher fungi consumed half of the amount of hemicellulose (i.e. 15%), leaving cellulose fairly intact, which should remain as an energy source for ruminants.     

The effect of white-rot basidiomycetes on chemical composition andin Vitro digestibility of wheat straw

Five white-rot basidiomycetes were evaluated for their potential to improve ruminal degradation of wheat straw.Polyorus brumalis, Lyophyllum ulmarium III,Trametes gibbosa, Pleurotus ostreatus, and aPleurotus ostreatus mutant were incubated on wheat straw for 30 d at 28°C. Detergent fiber, crude protein andin vitro dry matter digestibility (IVDMD) were determined. The results showed increasing crude protein and ash contents in fungus-treated straw. IVDMD values were increased in straws treated withP. ostreatus, P. ostreatus mutant andT. gibbosa only. Relative to untreated wheat straw the detergent fiber content—neutral detergent fiber (NDF), and acid detergent fiber (ADF) was reduced in fungus-treated straw and out of three fractions—hemicellulose, cellulose and lignin, hemicellulose showed the largest proportionate loss whereas lignin the smallest one in all 5 samples of fungus treated straw.     

Bioremediation of Polycyclic Aromatic Hydrocarbons in Creosote-Contaminated Soil by Peniophora incarnata KUC8836

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are present in products made from creosote, coal tar, and asphalt. When wood pile treated with creosote is placed in soil, PAHs can contaminate it. Creosote has been used for wood preservation in the past and is composed of approximately 85% PAHs and 15% phenolic compounds. PAHs cause harmful effects to humans and the environment because of their carcinogenic and mutagenic properties. White rot fungi can degrade not only lignin, but also recalcitrant organic compounds such as PAHs. Among numerous white rot fungi used in previous studies, four species were selected to degrade PAHs in a liquid medium. From this evaluation of the degradation of PAHs by the four fungal isolates, two species were ultimately selected for the highest rates of removal. Following 2 weeks of incubation with Peniophora incarnata KUC8836, the degradation rates of phenanthrene, fluoranthene, and pyrene were 86.5%, 77.4%, and 82.6%, respectively. Mycoaciella bispora KUC8201 showed the highest degradation rate for anthracene (61.8%). Hence, bioremediation of creosote-contaminated soil with an initial concentration of 229.49 mg kg^?1 PAHs was carried out using the two selected fungi because they could simultaneously degrade 13 more PAHs than the comparison species. More importantly, isolates of P. incarnata KUC8836 were discovered as powerful degraders of PAHs by producing laccase and manganese-dependent peroxidase (MnP), with 1.7- and 1.1-fold higher than the comparison species, respectively. Therefore, the white rot fungus may be proposed for the removal of PAHs and xenobiotic compounds in contaminated environments.     

Implementation and Monitoring of Soil Bioengineering Measures at a Landslide in the Middle Mountains of Nepal

Legume tissue quality is a key factor for enhancement of feed resources and contribution to soil fertility in mixed crop-livestock production systems. To compare methods used by soil scientists and animal-nutritionists to assess quality of plant materials, three woody tropical legumes with contrasting qualities were used: Indigofera zollingeriana Miq. (Indigofera), Cratylia argentea Benth. (Cratylia) and Calliandra houstoniana (Mill.) Stan. var. calothyrsus (Meiss.) Barn. CIAT 20400 (Calliandra). Plant material of each legume was used either fresh, freeze-dried, frozen, oven-dried (60 °C) or air-dried in order to estimate extents and rates of aerobic degradation in litterbags on the soil during 140 days and anaerobic degradation in an in-vitro gas production experiment during 144 h. Results showed, that aerobic decomposition rates of leaf tissues were highest for Indigofera (k = 0.013 day⁻¹), followed by Cratylia (k = 0.004 day⁻¹) and Calliandra (k = 0.002 day⁻¹). Gas production rates evaluated under anaerobic conditions, were highest for Indigofera (k = 0.086 h⁻¹), intermediate for Cratylia (k = 0.062 h⁻¹) and lowest for Calliandra (k = 0.025 h⁻¹). Decomposition and gas production rates differed (P < 0.001) among species. Differences between post harvest treatments were not statistically significant (P > 0.05). The extent of decomposition was highest for Indigofera (82.5%, w/w), followed by Cratylia (44.6%) and Calliandra (26.4%). The extent of gas production was highest for Indigofera (218.8 ml), followed by Cratylia (170.1ml) and Calliandra (80.1 ml). Extent of decomposition and extent of gas production were significantly different (P < 0.001) among species. In contrast to the extent of decomposition, the extent of gas production was affected (P < 0.001) by sample post harvest treatments. Highest gas production was observed for the fresh and frozen treatments. The forage quality parameters that best correlated with aerobic and anaerobic degradation were lignin+bound condensed tannins, lignin+total condensed tannins/N, indigestible acid detergent fibre (IADF) and in-vitro dry matter digestibility (IVDMD). Results showed that differences in decomposition and digestibility were more related to intrinsic plant quality parameters than to changes in tissue quality induced by post harvest treatments. In addition, we found that rate of aerobic degradation of legume leaves on the soil was highly correlated (r > 0.80, P < 0.001) to IVDMD and gas production (r = 0.53, P < 0.001). These results indicate that plant measurements (IADF, IVDMD and gas production) used to assess forage quality in animal nutrition studies are more rapid and resource saving predictors for aerobic decomposition of tropical legumes than initial plant quality ratios (lignin+polyphenols/N and lignin+total condensed tannins/N) commonly used by many researchers. Furthermore, this study confirms the potential usefulness of IVDMD for screening tropical legumes for soil fertility management.     

Influence of urea–calcium mixtures as rumen slow-release feed on in vitro fermentation using a gas production technique

In this experiment the effects of different urea products (urea [U] and urea–calcium mixtures [UCM]) on rumen fermentation were investigated in dependence of different energy sources by using in vitro techniques. The 7 × 2 factorial arrangement followed a completely randomised design using seven urea products (U100, U40CaCl₂, U50CaCl₂, U60CaCl₂, U40CaSO₄, U50CaSO₄ and U60CaSO₄) in combination with cassava chips (CC) or corn meal (CM). Compared with other treatments, the cumulative gas production (96 h) was significantly increased for U60CaCl₂ + CC and U60CaSO₄ + CC (p < 0.01), which was combined with a higher in vitro true digestibility (p < 0.01). In addition, the concentration of volatile fatty acids in the fluid of U60CaCl₂ + CC and U60CaSO₄ + CC was significantly higher than in other treatments. Urea treatments (U100 + CC and U100 + CM) caused the highest concentration of ruminal ammonia nitrogen (p < 0.01), which was significantly decreased by all UCM products in combination with CC, but not with CM. The highest levels of total bacteria, Fibrobacter succinogenes and anaerobic fungi were found for treatment U60CaCl₂ + CC and U60CaSO₄ + CC (p < 0.05). The findings revealed that the utilisation of U60CaCl₂ and U60CaSO₄ in combination with cassava chips improved the ruminal fluid fermentation in terms of NH₃-N and volatile fatty acid concentration, digestibility of energy and increased the fibrobacter concentrations.     

Immunopotentiating Activity of the Water-soluble Lignin Rich Fraction Prepared from LEM—The Extract of the Solid Culture Medium of Lentinus edodes Mycelia—

The water-soluble lignin in LEM (the extract of the solid culture medium of Lentinus edodes mycelia) has been known to have antiviral and immunopotentiating activities in vivo and in vitro. The water-soluble lignin rich fraction (JLS-18) was prepared from LEM using ultrafiltration and hydrophobic column chromatography. JLS-18 showed about 70 times higher antiviral activity than LEM in vitro. JLS-18 activated the cytotoxicity of NK cells and macrophages, and activated T cells in vitro. JLS-18 also induced interleukin 6 (IL-6) secretion from human leukocytes infected with Sendai virus in vitro. These data showed that JLS-18, the water-soluble rich fraction of LEM, had antiviral and immunopotentiating activities.