Octavio Loera

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.

Improvement of xylanase production by a parasexual cross between Aspergillus niger strains

A diploid strain (D4) isolated via parasexual recombination between two Aspergillus niger xylanase overproducing mutants was characterised in terms of enzyme production and catabolite repression by glucose. This strain increased xylanase production (607 nkat/ml), which was nearly 100% higher than titers achieved by the wild type strain (305 nkat/ml) and 28% higher than the best mutant used to induce parasexual cycle. Diploid D4 was also less sensitive to carbon catabolite repression by glucose, since xylanolytic activity was detected under conditions normally repressing production by the wild type strain. No decrease in maximal xylanase levels was observed in the presence of glucose for diploid D4.

Selection and Identification of Fungi Isolated from Sugarcane Bagasse and their Application for Phenanthrene Removal from Soil

This work investigated the identification and selection of fungi isolated from sugarcane bagasse and their application for phenanthrene (Phe) removal from soil. Fungi were identified by PCR amplification of ITS regions as Aspergillus terrus, Aspergillus fumigatus and Aspergillus niger, Penicillium glabrum and Cladosporium cladosporioides. A primary selection of fungi was accomplished in plate, considering Phe tolerance of every strain in two different media: potato dextrose agar (PDA) and mineral medium (MM). The radial extension rate (rr) in PDA exhibited significant differences (p < 0.05) at 200 and 400 ppm of Phe. A secondary selection of A. niger, C. cladosporoides, and P. glabrum sp. was achieved based on their tolerance to 200, 400, 600 and 800 ppm of Phe, in solid culture at a sugarcane bagasse/contaminated soil ratio of 95:5, in Toyamas, Czapeck and Wunder media. Under these conditions, a maximum (70%) Phe removal by A. niger was obtained. In addition C. cladosporioides and A. niger were able to remove high (800 ppm) Phe concentrations.

Pectinase production by a diploid construct from two Aspergillus niger overproducing mutants

Arginine auxotrophy (Arg 2 ) was induced in two Aspergillus nigerstrains, previously reported as 2-deoxy- D-glucose (2DG) resistant and pectinase overproducing mutants in either solid state fermentation (SSF) or submerged fermentation (SmF). Only one of these two arginine auxotrophs (strain Aw99ARG) was transformed with a plasmid containing the A. nidulands argB gene, indicating that this strain was affected in this particular gene. A diploid (D4) was generated via parasexual recombination between these two Arg 2 strains and was characterized with regard to 2DG sensitivity and pectinase production. This diploid showed wild type sensitivity to 2DG, suggesting that recessive mutations at two different loci were involved in 2DG resistance in each parent strain. The diploid had also increased pectinase production in SmF (127% higher) when compared to the wild type strain, from which all pectinase overproducing mutants were previously isolated, and 10% over Aw99-iii2 strain, formerly characterized as an overproducing strain in SmF.

Effect of moisture content and inoculum on the growth and conidia production by Beauveria bassiana on wheat bran

ABSTRACT The aim of the present work was to study the effect of moisture content and inoculum on the growth and conidia production by Beauveria bassiana on wheat bran (WB).The highest growth rate of B. bassiana 885.2 on WB media was obtained at a w =1.0, with no detected growth at a w < 0.97. Solid-state fermentation (SSF) using WB (66% moisture; a w =1.0) achieved a maximal yield of 1.18x10 10 conidia per gram of dry substrate (gds). This yield decreased one order of magnitude with higher moisture contents or the addition of sugarcane bagasse (SCB) as a texturiser. In SSF using WB (66% humidity),the time to obtain a yield of 1x10 10 conidia/gds, referred to as t 10 , could be predicted using a model considering common inoculum levels and maximal yields. For instance, t 10 was 285 h with an inoculum of 1x10 6 conidia/gds; however, t 10 was reduced to 232 h and 148 for inocula of 7x10 6 and 5x10 7 conidia/gds, respectively. The estimation of t 10 values allowed both comparison between the cultures and prediction of harvesting times in production processes. Values for hydrophobicity were within 90 and 92%, whereas viability averages were around 70% for all the cultures Key words

Differential patterns of constitutive intracellular laccases of the vegetative phase of Pleurotus species

The constitutive intracellular laccase activity of ten strains of Pleurotus spp. was determined in vitro and by zymograms, using different substrates. Differences in the in vitro activities were observed between all the strains; however, zymogram patterns were only similar for strains within same species, independently of any of the three substrate (2,6-dimethoxyphenol, p-anisidine or o-tolidine) used. The differences observed in the number and positions of the isoforms in the gel suggest that laccase zymograms can be used to differentiate species of this organism.

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination.

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.

Oxygen-rich culture conditions enhance the conidial infectivity and the quality of two strains of Isaria fumosorosea for potentially improved biocontrol processes

BACKGROUND In addition to high production levels of conidia, the success of entomopathogenic fungi as biological control agents depends both on their prevalence under the environmental conditions found in open fields (resistance to stress) and on the capacity of these conidia to infect pests. This study compares conidium production, infectivity and resistance to thermal and osmotic stress in two strains of Isaria fumosorosea (ARSEF 3302 and CNRCB1) grown either under a normal atmosphere (21% O2) or using enriched oxygen pulses (26% O2). RESULTS After 180 h, the ARSEF 3302 strain with 26% O2 pulses increased conidium production nearly fivefold compared with the normal atmosphere, while conidium production by the CNRCB1 strain decreased by 50% under O2 pulses, relative to the values measured with the normal atmosphere. The conidia obtained with 26% O2 pulses had a greater germination rate and resistance to thermal and osmotic stress, in addition to improved infectivity against Galleria mellonella (Lepidoptera) larvae. These findings were associated with an increase in catalase activities for both strains. CONCLUSION An enriched oxygen atmosphere increases the quality of conidia of both strains of I. fumosorosea, with a variable effect on conidium production.