Carmen Sánchez

Effect of substrate particle size and additional nitrogen source on production of lignocellulolytic enzymes by Pleurotus ostreatus strains.

Two strains of Pleurotus ostreatus (IE-8 and CP-50) were grown on defined medium added with wheat straw extract (WSE). Mycelia from these cultures were used as an inoculum for solid fermentation using sugar cane bagasse (C:N=142). This substrate was used separately either as a mixture of heterogeneous particle sizes (average size 2.9 mm) or as batches with two different particle sizes (0.92 mm and 1.68 mm). Protein enrichment and production of lignocellulolytic enzymes on each particle size was compared. The effect of ammonium sulphate (AS) addition was also analyzed (modified C:N=20), this compound favored higher levels of protein content. Strain CP-50 showed the highest increase of protein content (48% on particle size of 1.68 mm) when compared to media with no additional N source. However, strain IE-8 produced the highest levels of all enzymes: xylanases (5.79 IU/g dry wt on heterogeneous particles) and cellulases (0.18 IU/g dry wt on smallest particles), both without the addition of AS. The highest laccase activity (0.040 IU/g dry wt) was obtained on particles of 1.68 mm in the presence of AS. Since effect of particle size and addition AS was different for each strain, these criteria should be considered for diverse biotechnological applications.

Laccases of Pleurotus ostreatus observed at different phases of its growth in submerged fermentation: production of a novel laccase isoform

The production of laccases during the lag, exponential and stationary phases of growth of Pleurotus ostreatus in submerged fermentation was evaluated. Laccase activity was positively correlated to the growth of the fungus. The specific growth rate was 0.02 h(-1) and the highest amount of dry biomass (7.8 gl(-1)) was obtained after 480 h of growth. Four laccase isoforms were secreted by the fungus, and tentatively named L(I)1, L(I)2, L(I)3 and L(I)4. L(I)2, L(I)3 and L(I)4 were produced during the stationary phase (between 408 and 456 h approximately) while L(I)1 was produced during the lag, exponential and stationary phases of growth. Maximal laccase activity (12 200 Ul(-1)) was observed at the beginning of the stationary phase (at 432 h of growth). L(I)1 was purified by preparative isoelectric focusing and partially characterized. L(I)1 had a molecular mass of 43.7 kDa as determined by SDS PAGE, a Km and Vmax of 90 microM and 1.18 DeltaAbs min(-1) respectively and an isoelectric point of 2.3. L(I)1 showed activity over a broad range of pH and temperature, which may make it useful in the biodegradation of phenolic compounds present in wastewater from several industrial processes.

Particle geometry affects differentially substrate composition and enzyme profiles by Pleurotus ostreatus growing on sugar cane bagasse

The growth of Pleurotus ostreatus was analyzed on three particle sizes of sugar cane bagasse: 0.92 mm and 1.68 mm in diameter, in addition to heterogeneous fibers (average 2.9 mm in diameter). Specific growth rate on heterogeneous particles was lower (μ=0.043 h(-1)), although soluble protein production was maximal (809 μg/g dry wt). Higher μ values were reached on the other two particles sizes (0.049-0.05 h(-1)) with less soluble protein (500 μg/g dry wt). Xylanases and laccases were favored in heterogeneous particles; while the highest selectivity for xylanases over cellulases was observed in 1.68 mm particles, corresponding with the maximal hemicellulose breakdown. Lignin and cellulose were preferentially degraded in smallest particles. This study shows that the geometrical ratio, shape and size of sugar cane bagasse fibers strongly influence packing density for SSF substrate, with an impact in the production of extracellular enzymes, growth rates and composition changes in substrate.

Detection of highly productive strains of Pleurotus ostreatus by their tolerance to 2-deoxy-D-glucose in starch-based media

An identification procedure for fast fructifying strains of Pleurotus ostreatus was developed using maize starch as carbon source, mineral salts and 0·1 g l −1 2-deoxy- d -glucose (2-DG). Three strains that grew on Petri dishes in these conditions were called 2-DG tolerant. In trials under normal farming conditions, tolerant strains fruited an average of 19 days faster and had a mean productivity (g daily yield of edible mushrooms kg −1 of compost raw material) which was twice that of three other strains that were 2-DG sensitive in growth tests in vitro . Analysis of mycelial morphology indicated that with starch as main carbon source increasing concentrations of 2-DG reduced the hyphal diameter ( D h ) and length of distal hyphal segments ( L av ) in all strains. On media containing glucose as main carbon source the D h of the tolerant strains was only 60% of that of the sensitives, and the D h of tolerant strains increased an average 53 % with addition of 0·01 g l −1 of 2-DG. The hyphal diameter of sensitive strains tended to decrease when 2-DG was added to the glucose-based culture medium. L av showed no regular pattern among strains. The 2-DG tolerant phenotype in maize starch medium may be a useful phenotype for selection of highly productive strains of P. ostreatus for commercial exploitation. Its physiological basis is unclear, but the observed changes in hyphal diameter suggest that sublethal concentrations of the glucose analogue may stimulate mycelial growth of tolerant strains in some way.

Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum: Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the manufacture of plastics, and it is an environmental contaminant. The specific growth rate (μ), maximum biomass (Xmax), biodegradation constant of DEHP (k), half-life (t1/2) of DEHP biodegradation and removal efficiency of DEHP, esterase and laccase specific activities, and enzymatic yield parameters were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000mg/L). The greatest μ and the largest Xmax occurred in media supplemented with 1000mg of DEHP/L. F. culmorum degraded 95% of the highest amount of DEHP tested (1000mg/L) within 60h of growth. The k and t1/2 were 0.024h(-1) and 28h, respectively, for both DEHP concentrations. The removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500mg/L, respectively. Much higher specific esterase activity than specific laccase activity was observed in all media tested. The compounds of biodegradation of DEHP were identified by GC-MS. A DEHP biodegradation pathway by F. culmorum was proposed on the basis of the intermolecular flow of electrons of the identified intermediate compounds using quantum chemical modeling. DEHP was fully metabolized by F. culmorum with butanediol as the final product. This fungus offers great potential in bioremediation of environments polluted with DEHP.

In the midst of death we are in life: Further advances in the study of higher fungi

Summary Senescence and death of mushroom fruit bodies are the natural terminations of their development but not the end of their life because a new batch of fruit bodies can arise directly from the old. A fungal version of programmed cell death seems to be involved in sculpturing fruit body shapes. Fungal differentiation appears to be quite flexible and greatly influenced by the immediate microenvironment. Techniques are now emerging which will enable the earliest stages in fruit body initiation to be studied.

Conventional histological stains selectively stain fruit body initials of basidiomycetes

A method to identify the earliest stages (initials) of Pleurotus pulmonarius and Coprinus cinereus fruit body formation was developed. Light microscopy and Cryo-SEM were used to confirm that even the smallest structures to take up stain were initials of fruit bodies. An approach combining histological staining (flooding the Petri dish with 1% toluidine blue in 1% boric acid (w/v) for 15 min) and image analysis allowed the number of fruit bodies formed on Petri dishes to be quantified easily. Use of the vital stain Janus green (0.001% aqueous, w/v) allowed continued observation of living tissue so that the proportion of fruit bodies that matured (30%) could be established. The method was also effective on wheat straw cultures and could be used to monitor development of mature fruit bodies. It is a promising tool in the study of physiological processes involved in fruit body initiation.

A novel biodegradation pathway of the endocrine-disruptor di(2-ethyl hexyl) phthalate by Pleurotus ostreatus based on quantum chemical investigation

Di(2-ethyl hexyl) phthalate (DEHP) is a plasticizer that interfere with endocrine systems in mammals. Growth parameters for Pleurotus ostreatus grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000mg/L) were evaluated. The highest biomass production was observed in medium supplemented with 1000mg of DEHP/L. Half-life of DEHP biodegradation, biodegradation constant of DEHP, and percentage of removal efficiency (%E) were also determined. P. ostreatus degraded 100% of DEHP after 504h. %E was 99.3% and 98.4% for 500 and 1000mg of DEHP/L, respectively. Intermediate compounds of biodegraded DEHP were identified by GC-MS and a DEHP biodegradation pathway was proposed using quantum chemical investigation. DEHP might be metabolized through three pathways; a de-esterification pathway, an oxidation pathway and an oxidation-hydrolysis pathway, forming phthalic acid, acetic acid and butanediol, respectively. P. ostreatus degrades and uses (as carbon and energy source) high concentrations of DEHP.

Microscopic observations of the early development of Pleurotus pulmonarius fruit bodies

From observations made by light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy we conclude that the expansion of the young fruit body of Pleurotus pulmonarius involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix (ECM), the components of which must be under the control of the hyphae which the ECM surrounds. However the ECM in these fruit bodies is a dilute material. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae. Observations of the hyphal and fruit body structures with a range of conventional microscopic techniques are crucial to complement the information obtained through physiological and molecular studies for understanding the cellular changes that occur during mushroom development.

Characterization of the Solid-State and Liquid Fermentation for the Production of Laccases of Pleurotus ostreatus

In this chapter, the activity and isoenzymes number of laccases of Pleurotus ostreatus grown in solid-state and liquid fermentations are reported. An atypical behavior of this fungus with relation on enzyme production was observed, since the major laccase activity levels were observed in liquid fermentation, whereas the solid-state fermentation has been recognized as better system for enzyme production.