White-rot fungal growth on sugarcane lignocellulosic residue

Summary Twelve white-rot fungi were grown in solid state culture on sugarcane chips previously fermented by yeast employing the EX-FERM process. The lignocellulosic sugarcane residue had 12.5% permanganate lignin and 81.3% holocellulose. After 5 to 6 weeks at 20° C, all fungi produced a solid residue which had a lower in vitro dry matter enzymatic digestibility than the original bagasse, with the exception of Coriolus versicolor which showed a slight increase of 0.6 units. Four fungi produced a residue with higher soluble solids than the original sample. Lignin losses were rather similar for all fungi tested, an average value of 38.64% of the original value was obtained. About the same amount of hemicellulose was degreaded, 32.22%. Most fungi showed a preference for hemicellulose hydrolysis over cellulose degradation. The two fungi that showed greater cellulolytic activity were Sporotrichum pulverulentum and Dichomitus squalens. No appreciable dry matter losses were detected for Agrocybe aergerita and Flammulina velutipes.     

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.     

Microbial Delignification with White Rot Fungi Improves Forage Digestibility

Three wild-type white rot fungi and two cellulase-less mutants developed from Phanerochaete chrysosporium K-3 (formerly Sporotrichum pulverulentum) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy. P. chrysosporium K-3 and Ceriporiopsis subvermispora FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However, C. subvermispora removed the most lignin and significantly improved biodegradation over all other treatments. Phellinus pini RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from P. chrysosporium K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that C. subvermispora extensively removed ester-linked p-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that C. subvermispora removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues). C. subvermispora improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.     

Treatment of oral thrush in HIV/AIDS patients with lemon juice and lemon grass (Cymbopogon citratus) and gentian violet

PurposeThe purpose of the study was to investigate the safety and efficacy of lemon juice and lemon grass (Cymbopogon citratus) in the treatment of oral thrush in HIV/AIDS patients when compared with the control group using gentian violet aqueous solution 0.5%. Oral thrush is a frequent complication of HIV infection.In the Moretele Hospice, due to financial constraints, the treatment routinely given to patients with oral thrush is either lemon juice directly into the mouth or a lemon grass infusion made from lemon grass (Cymbopogon citratus) grown and dried at the hospice. These two remedies have been found to be very efficacious therefore are used extensively. Gentian violet, the first line medication for oral thrush in South Africa, is not preferred by the primary health clinic patients due to the visible purple stain which leads them to being stigmatized as HIV-positive. Cymbopogon citratus and Citrus limon have known antifungal properties.MethodsThe study design was a randomised controlled trial. Ninety patients were randomly assigned to one of three groups: gentian violet, lemon juice or lemon grass. Inclusion criteria included being HIV-positive with a diagnosis of oral thrush. The study period was 11 days and patients were followed up every second day. International ethical principles were adhered to during the study.ResultsOf the 90 patients, 83 completed the study. In the intention-to-treat analysis, none of the p-values were significant therefore the null hypothesis could not be rejected. In the analysis of the participants who actually completed the trial, the lemon juice showed better results than the gentian violet aqueous solution 0.5% in the treatment of oral thrush in an HIV-positive population (p<0.02). The null hypothesis in terms of the lemon grass and gentian violet could also be rejected on the basis of the Chi-square test and the likelihood ratio test (p<0.05).ConclusionsThough the patient population was small, the use of lemon juice and lemon grass for the treatment of oral candidiasis in an HIV population was validated by the randomised controlled trial.     

Effect of six species of white-rot basidiomycetes on the chemical composition and rumen degradability of wheat straw

This study was conducted to investigate changes in in vitro dry matter digestibility (IVDMD), volatile fatty acids (VFA) production and cell-wall constituent degradation in wheat straw treated with six white-rot fungi: Daedalea quercina, Hericium clathroides, Phelinus laevigatus, Inonotus andersonii, Inonotus obliquus, and Inonotus dryophilus. The incubation of wheat straw for 30 days at 28 C improved IVDMD from 41.4 (control) to 59.2% for D. quercina, 56.3% for H. clathroides, 50.2% for P. laevigatus, 51.4% for I. andersonii, 52% for I. obliquus, and 55.9% for I. dryophilus. In contrast, the growth of fungi was accompanied by the dry matter loss of wheat straw: 43% for D. quercina, 12% for H. clathroides, and 22-25% for the other fungi. It is evident that the increase in digestibility by D. quercina was not offset by a loss of dry matter. The total VFA production during the rumen fermentation of fungus-treated straw was slightly increased by H. clathroides and I. dryophilus only. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were reduced in fungus-treated straw. Out of the three fractions (hemicellulose, cellulose, and lignin), hemicellulose and lignin showed the largest proportionate loss after inoculation with the fungi D. quercina, H. clathroides, P. laevigatus, and I. obliquus. The other two fungi showed the largest proportionate loss in cellulose and hemicellulose contents. The results of this study suggest that the digestion enhancement of wheat straw colonized by white-rot fungi is regulated by complex factors including the degradation of structural carbohydrates and lignin.     

Biodegradation of paddy straw obtained from different geographic locations by means of <Emphasis Type="Italic">Phlebia</Emphasis> spp. for animal feed

Various cereal straws are used as feed by supplementing the green forage or other feed stuffs. An experiment was designed to see the effect of different geographic locations and climatological conditions on biochemical constituents, fungal degradation and in vitro digestibility of paddy straw. Paddy straw (PS) obtained from three different geographic locations of India was subjected to solid state fermentation using four white rot fungi i.e. Phlebia brevispora, P. fascicularia, P. floridensis and P. radiata. Changes in the biochemical constituents like water soluble content, hemicellulose, cellulose, lignin, total organic matter, and in vitro digestibility of paddy straw was analyzed over a period of 60 days along with lignocellulolytic enzymes i.e. laccase, xylanase and carboxymethyl cellulase. All the fungi degraded the straw samples and enhanced the in vitro digestibility. The paddy straw, obtained from north western zone (NWZ) suffered a maximum loss (228 g/kg) of lignin by P. radiata, while a maximum enhancement of in vitro digestibility from 185 to 256 g/kg was achieved by P. brevispora, which also caused minimum loss in total organic matter (98 g/kg). In PS obtained from central eastern zone (CEZ) and north eastern zone (NEZ), a maximum amount of lignin (210 and 195 g/kg, respectively) was degraded by P. floridensis and resulted into a respective enhancement of in vitro digestibility from 172 to 246 g/kg and 188 to 264 g/kg. The study demonstrates that geographic locations not only affect the biochemical constituents of paddy straw but the fungal degradation of fibers, their in vitro digestibility and lignocellulolytic enzyme activity of the fungus may also vary.     

Biodelignification of wheat straw and its effect on in vitro digestibility and antioxidant properties

A variety of methods for feed development have been introduced during last few years. Bioprocessed agricultural residues may prove a better alternative to provide animal feed. For the purpose, some white rot fungi were allowed to degrade wheat straw up to 30 days under solid state conditions. Several parameters including loss in total organic matter, ligninolysis, in vitro digestibility of wheat straw and estimation of different antioxidant activities were studied. All the fungi were able to degrade lignin and enhance the in vitro digestibility. Among all the tested fungi, Phlebia brevispora degraded maximum lignin (30.6%) and enhanced the digestibility from 172 to 287 g/kg. Different antioxidant properties of fungal degraded wheat straw were higher as compared to the uninoculated control straw. Phlebia floridensis found to be more efficient organism in terms of higher antioxidant activity (70.8%) and total phenolic content (9.8 mg/ml). Thus, bioprocessing of the wheat straw with the help of these organisms seems to be a better approach for providing the animal feed in terms of enhanced digestibility, higher protein content, higher antioxidant activity and availability of biomass.     

Selection of white-rot basidiomycetes for bioconversion of mustard (Brassica compestris) straw under solid-state fermentation into energy substrate for rumen micro-organism

Aims: Selection of white‐rot fungi of bio‐conversion of mustard straw (MS) into feed for ruminants. Methods and Results: Mustard straw was cultured with Ganoderma applanatum, Coriolus versicolor and Phanerochaete chrysosporium for solid‐state fermentation at 35°C from 7 to 63 days for dilignification and for 21 days to study dry matter digestibility and protein enrichment. Lignin loss in fungus cultured straw varied between 100 and 470 g kg^?1 lignin. Dilignification was higher between 7 and 28 days fermentation with C. versicolor. Among the three fungi P. chrysosporium was the most effective in degrading lignin for longer fermentation. In‐vitro dry matter digestibility (IVDMD) and crude protein content was higher in C. versicolor cultured straw. Large quantity of straw was cultured by C. versicolor for 21 days, for in vivo evaluation. Mean pH and metabolites of rumen fermentation were not different while, pH and volatile fatty acid increased at 6 h postfermentation on cultured straw feeding. Cultured straw fermentation increased (P = 0·001) small holotricks and reduced (P = 0·005) large holotricks population. Fungus cultures straw did not improve microbial enzyme concentration. Conclusions: Coriolus versicolor and P. chrysosporium were the promising fungus for MS bio‐dilignification. Significance and Impact of the Study: Coriolus versicolor treated MS improved dry matter digestibility and protein content.     

Radiation and fermentation treatment of cellulosic wastes

The effect of radiation pasteurization of sugar cane bagasse and rice straw and fermentation using various strains of fungi were studied for upgrading of cellulosic wastes. The initial contamination by fungi and aerobic bacteria both in bagasse and straw was high. The doses of 30 kGy for sterilization and 8 kGy for elimination of fungi were required. Irradiation effect showed that rice straw contained comparatively radioresistant microorganisms. It was observed that all the fungi (Hericium erinacium, Pleurotus djamor, Ganoderma lucidum, Auricularia auricula, Lentinus sajor-caju, Coriolus versicolor, Polyporus arcularius, Coprinus cinereus) grow extending over the entire substrates during one month after inoculation in irradiated bagasse and rice straw with 3% rice bran and 65% moisture content incubated at 30°C. Initially, sugar cane bagasse and rice straw substrates contained 39.4% and 25.9% of cellulose, 22.9% and 26.9% of hemicellulose, and 19.6% and 13.9% of lignin + cutin, respectively. Neutral detergent fibre (NDF) values decreased significantly in sugar cane bagasse fermented byG. lucidum, A. auricula andP. arcularius, and in rice straw fermented by all the 8 strains of fungi. Acid detergent fibre (ADF) values also decreased in bagasse and rice straw fermented by all the fungi.P. arcularius, H. erinacium, G. lucidum andC. cinereus were found to be the most effective strains for delignification of sugar cane bagasse.     

Rumen Fungi and Forage Fiber Degradation

The role of anaerobic rumen fungi in in vitro forage fiber degradation was determined in a two forage × two inoculum source × five treatment factorial design. Forages used as substrates for rumen microorganisms were Coastal bermuda grass and alfalfa; inoculum sources were rumen fluid samples from a steer fed Coastal bermuda grass hay or alfalfa hay; treatments were whole rumen fluid (WRF), WRF plus streptomycin (0.2 mg/ml of rumen fluid) and penicillin (1.25 mg/ml of fluid), WRF plus cycloheximide (0.5 mg/ml of fluid), WRF plus streptomycin, penicillin, and cycloheximide, and McDougall buffer. Populations of fungi as shown by sporangial development were greater on bermuda grass leaves than on alfalfa leaflets regardless of inoculum source. However, endogenous fungal populations were greater from the alfalfa hay inoculum. Cycloheximide inhibited the fungi, whereas streptomycin and penicillin, which inhibit bacterial populations, resulted in an increase in numbers of sporangia in the alfalfa inoculum, suggesting an interaction between bacteria and fungi. Bacteria (i.e., WRF plus cycloheximide) were equal to the total population in degrading dry matter, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and cellulose for both inocula and both forages. Degradation of dry matter, NDF, ADF, and cellulose by anaerobic fungi (i.e., WRF plus streptomycin and penicillin) was less than that due to the total population or bacteria alone. However, NDF, ADF, and cellulose digestion was 1.3, 2.4, and 7.9 percentage units higher, respectively, for bermuda grass substrate with the alfalfa versus bermuda grass inoculum, suggesting a slight benefit by rumen fungi. No substantial loss of lignin (72% H₂SO₄ method) occurred due to fungal degradation. The most active fiber-digesting population in the rumen was the bacteria, even when streptomycin and penicillin treatment resulted in an increase in rumen fungi over untreated WRF. The development of large numbers of sporangia on fiber may not indicate a substantial role as digesters of forage.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Prediction of the day matter digestibility of tropical grasses using a pepsin-cellulase assay

A study was made of the accuracy of predicting dry matter digestibility in vivo from the dry matter disappearance of forage samples in pepsin (48 h) followed by Onozuka cellulase (48 h). Forty-five samples representing four tropical pasture species, Cenchrus ciliaris, Chloris gayana, Digitaria spp., Setaria spp. and one temperate grass, Lolium perenne were used.Dry matter digestibility in vivo was significantly correlated with the disappearance in pepsin-cellulase (r = 0.94) and the regression predicting dry matter digestibility in vivo had a residual standard deviation of ± 2.7 digestibility units. Separate regressions for the five grasses had RSD values ranging from ± 1.3 to ± 2.6.It was concluded that the digestibility in vivo of both tropical and temperate grasses in the range 40–76% digestibility could be accurately predicted by the pepsin-cellulase assay, provided samples of known digestibility in vivo similar to those being tested were included as standards in each run.     

Fermentation characteristics and feeding value of ensiled poultry litter containing wheat straw,bagasse or sawdust

Poultry excreta substantially increased the crude protein (CP) content and the calcium and phosporus content of the base bedding materials. The crude fibre (CF) content of sawdust (52.3%) was higher than that of wheat straw (38.9%) and bagasse (30.2%). Dry matter digestibility in vitro (IVDMD) of wheat straw, bagasse and sawdust poultry litters (PL) was 65.4, 64.5 and 48.1%, respectively. Green sorghum fodder when ensiled alone or with 20% wheat straw PL, sawdust PL or bagasse PL on fresh basis contained 4.67, 7.80, 10.00 and 7.55% CP, respectively. Nitrogen-free extract (NFE) content of PL silages was lower than that of the control. Apart from wheat straw PL, all silages accumulated considerable amounts of lactic acid. The total volatile fatty acids (TVFA) concentrations were similar for all silages. The addition of PL caused an increase in the proportion of ammonia nitrogen. A feeding trial with crossbred adult male cattle revealed no significant difference in dry matter (DM), CP and ether extract (EE) digestibility of wheat straw and bagasse PL silages. The CF digestibility was similar for all the silages. Sawdust PL silage, however, was significantly lower (P < 0.05) in digestibility of DM, EE and NFE compared to other PL based silages. The DCP and TDN values for the control, wheat straw, bagasse and sawdust PL silages were 2.0, 60.1; 4.3, 45.3; 6.1, 50.3 and 2.9, 41.9 kg/100 kg DM, respectively.     

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.     

Carbon dioxide compensation values in citronella and lemongrass

Carbon dioxide compensation values of mature leaves from 10 selections of citronella (Cymbopogon nardus [L.] Rendle) grown at 32/27 or 27/21 C day/night temperatures and three strains of lemongrass (Cymbopogon citratus [D.C.] Stapf. and Cymbopogon flexuosus [D.C.] Stapf.) grown at 8- or 15-hour photoperiods were measured in a controlled environment at 25 C. All leaves had low compensation values but citronella varied from 1.3 to 9.7 μl/liter and lemongrass from 0.7 to 3.5 μl/liter. Lower growing temperature generally resulted in lower compensation values for citronella but there was no consistent photoperiod effect on lemongrass.     

Enzymological Characterization of Atm,the First Laccase from Agrobacterium sp. S5-1, with the Ability to Enhance In Vitro digestibility of Maize Straw

Laccase is an enzyme that catalyzes oxidation of phenolic compounds, diamines and aromatic amines. In this study, a novel laccase-like gene (atm) in a ligninolyitic isolate Agrobacterium sp. S5-1 from soil humus was identified and heterologously expressed in Escherichia coli. Atm exhibited its maximal activity at pH 4.5 and at 50°C. This enzyme was tolerant to high temperature, a broad range of pH, heavy metal ions (Co³⁺, Mn²⁺, Cu²⁺ and Ni²⁺, 20 mM) and all tested organic solvents. Furthermore, Atm significantly (p<0.05) increased dry matter digestibility of maize straw from 23.44% to 27.96% and from 29.53% to 37.10% after 8 or 24 h of digestion and improved acid detergent fiber digestibility from 5.81% to 10.33% and from 12.80% to 19.07% after 8 or 24 h of digestion, respectively. The combination of Atm and fibrolytic enzymes significantly (p<0.05) enhanced neutral detergent fiber digestibility from 19.02% to 24.55% after 24 h of digestion respectively. Results showed treatment with Atm effectively improved in vitro digestibility of maize straw, thus suggesting that Atm has an application potential for bioconversion of lignin rich agricultural byproducts into animal feed and cellulosic ethanol.     

Inhibitory effect of lemongrass oil and its major constituents on Candida biofilm and germ tube formation

Adhesion to a variety of host cells and the surface of biomaterials is a critical step in successful colonization and infection by Candida spp. Several essential oils are known to possess antifungal properties and are potentially used as antifungal agents. By studying the efficacy of essential oils against different pathogenic fungi in the genus Candida, we have evaluated the in vitro antifungal effects of eight essential oils used in aromatherapy, namely holy basil (Ocimum sactum L), lemongrass (Cymbopogon citratus DC), citronella grass (Cymbopogon winterianus Jowitt), kaffir lime (Citrus hystrix DC), sweet basil (Ocimum basilicum Linn), Plai (Zingiber cassumunar Roxb), Curcuma (Curcuma longa Linn), and ginger (Zingiber officinale Rose), against Candida albicans and Candida krusei in both planktonic and biofilm form. The results revealed that among the tested essential oils, lemongrass oil exhibited the most effective killing activity and possessed the strongest inhibitory effect on Candida biofilm formation. In addition, lemongrass oil and its major constituents can inhibit germ tube formation, which might affect adherence. The data in this study indicates that lemongrass oil possessed antibiofilm activity and could modulate candidal colonization. Therefore, it is a promising essential oil to combat candidal colonization and infection.     

Botanical composition and extent of lignification affecting digestibility of wheat and oat straw and paspalum hay

Samples of 1 kg of wheat straw, oat straw and paspalum hay were separated manually into botanical fractions, and the three largest fractions of each forage were analysed chemically for cell-wall constituents, silica and nitrogen. Proportions of digested dry matter, cellulose and hemicellulose, and potential digestibility for each of these major botanical fractions were determined when chaffed samples were placed in nylon bags and incubated for 12, 24, 48, 72 and 96 h in the rumen of sheep fed on lucerno. Cross-sections of botanical fractions were stained with safranin and fast green, and proportions of lignified tissue determined by light-microscopy and planimetry.Large differences in dry-matter digestibility between wheat straw and oat straw were attributed to the different proportions of botanical fractions. Within forages, stem was the largest fraction, the most lignified and had the lowest potential digestibility. Proportions of digested dry matter from botanical fractions at 12 h were poorly correlated with lignin content of dry matter (r = 0.25) but at 72 h were negatively correlated with lignin content of dry matter (r = ?0.84, P<0.01) and with proportions of lignified tissue (r = ?0.67, P<0.05) in the respective botanical fractions. Proportions of cellulose and hemicellulose digested at 72 h were strongly correlated with lignin content of cell walls (r = ?0.90, P<0.01; r = ?0.85, P<0.01, respectively). Proportions of lignified tissues were less closely correlated with all measurements of digestibility than were proportions of lignin in cell walls determined chemically. Development of a technique for measuring intensity of lignification might enhance the value of light-microscopy measurements.     

Digestibility of a high-fiber biscuit-based diet by black and white colobus (Colobus guereza)

The acceptability and digestibility of a high-fiber biscuit-based diet was investigated using two adult male Colobus guereza animals. Although the animals were initially reluctant to accept the biscuit, it was eventually readily consumed. Apparent digestion coefficients for the diet (average composition, dry matter basis: 16% crude protein, 25% neutral detergent fiber (NDF), 9.5% acid detergent fiber (ADF), 1.2% acid lignin) determined by total fecal collection were 0.871 for dry matter, 0.813 for NDF, 0.693 for ADF, and 0.208 for acid lignin. Fiber digestive capabilities in C. guereza generally exceeded those reported in ruminant species based on predictive equations. Use of acid lignin and Cr₂O₃ as markers underestimated dry matter digestibility by 3.9 and 6.0%, respectively.     

Metabolisable energy of grass and red clover silages fed to sheep at maintenance level

This study investigated the effect of forage type (grass or red clover) and harvesting time (primary growth or regrowth) of silage on energy and N utilisation by sheep fed at maintenance level. Specifically, the assumption of constant loss of energy of digestible organic matter from energy losses in urine and CH₄ applied in evaluation of silage metabolisable energy (ME) was investigated. Urinary excretion of high-energy phenolic compounds related to solubilisation of lignin was assumed to affect urinary energy (UE) losses from sheep fed highly digestible grass silage (GS). A total of 25 primary growth and regrowth silages of timothy (Phleum pratense) and meadow fescue (Festuca pratensis) grass mixtures and red clover (Trifolium pratense) samples collected in digestibility trials with sheep, including faecal and urine samples, were used for energy and N determinations. Urinary concentration of monophenolic compounds and CH₄ emissions in vitro were also analysed. Daily faecal N output, CH₄ yield (MJ/kg DM intake), proportion of CH₄ energy in digestible energy (DE) and proportion of UE in DE were greater (P ≤ 0.03) in sheep fed red clover silage (RCS) than GS. Furthermore, less (P = 0.01) energy was lost as UE of DE in sheep fed primary growth GS compared with the other treatments. The relationship between UE and silage N intake or urinary N output for both silage types (i.e. grass v. red clover) was strong, but the fit of the regressions was better for GS than RCS. The CH₄/DE ratio decreased (P < 0.05) and the UE/DE ratio increased (P < 0.05) with increasing organic matter digestibility in RCS. These relationships were not significant (P < 0.05) for the GS diets. The regression coefficient was higher (P < 0.05) for GS than RCS when regressing ME concentration on digestible organic matter. The results of this study imply that ME/DE ratio is not constant across first-cut GS of different maturities. The ME production response may be smaller from highly digestible first-cut GS but could not be clearly related to urinary excretion of monophenols derived from solubilisation of lignin. Furthermore, energy lost in urine was not clearly defined for RCS and was much more predictable for GS from silage N concentration.