Rice recycling: a simple strategy to improve conidia production in solid-state cultures.

Here we studied at a laboratory scale a potential strategy to revalorize the residual rice remaining at the end of a conventional conidia production process in solid-state culture. The conidia production of Trichoderma asperellum Th-T4 (3) and Metarhizium robertsii Xoch-8.1 started with the use of fresh rice (unrecycled rice) as the substrate (cycle one), and continued with the use of recycled rice in successive cycles of conidia production. The rice remaining at the end of the first cycle was reused without any further sterilization or reinoculation. As a result, it was observed that the conidia production and productivity significantly increased in both fungi. Conidia production in T. asperellum Th-T4 (3) increased from 1 × 109 (first cycle) to 2·9 × 109 conidia per gram of initial dry substrate (con⋅gds-1 ) (second cycle using recycled rice), while in M. robertsii Xoch-8.1, this parameter increased form 5·7 × 108 to 1·4 × 109 con⋅gds-1 . Both fungi grew faster and conidiated earlier when recycled rice was used as the substrate, therefore, conidia productivity was also significantly improved. Furthermore, the use of recycled rice did not affect conidia viability. This is the first report about a recycling methodology completely free of extra-processing steps, and useful to increase conidia production and productivity.

Improved conidiation from entomopathogenic fungi through 26% oxygen pulses in solid-state culture depends on a balance between headspace volume and substrate amounts.

Oxygen-enriched atmospheres applied as periodic pulses increased conidia production from entomopathogenic fungi in agar surface cultures. However, this advantage has not been obtained in solid-state cultures (SSC), probably as a result of different biomass production between both culture systems. In this work, the biomass formation from two Isaria strains was limited in SSC using 5, 2·5 and 1 initial grams of substrate (gds). In the system with 5 gds, conidia production decreased in 26% oxygen-enriched pulses compared to the normal atmosphere. Conversely, 26% oxygen pulses increased conidiation up to one order magnitude in systems with 2·5 and 1 gds, respective to the normal atmosphere. These results were explained by oxygen depletion and high CO2 accumulation in the 5 gds system. Whereas in systems with 2·5 or 1 gds, oxygen levels remained high enough to stimulate conidiation. These results were attributed to the headspace volume:gds ratio, which is suggested to be ≥48 ml per gds. This ratio is proposed as a scaling-up criterion for bioreactor design when oxygen-enriched pulses are used in SSC for improvement of conidia production. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxygen-enriched atmospheres applied as periodic pulses increase conidiation in entomopathogenic fungi (EF). However, this remained restricted to agar surface cultures, since conidiation decreased when carried out in solid-state culture (SSC) which is used as large-scale production system. We identified that in SSC the ratio between the headspace volume containing 26% oxygen-enriched pulses and the grams of substrate determines the conidiation response to oxygen-enriched pulses. For the first time, oxygen-enriched pulses increased conidiation in SSC respective to the normal atmosphere in four EF. This ratio is proposed as a bioreactor criterion design for large-scale conidia production of EF using oxygen-enriched pulses.

Reactive oxygen species production, induced by atmospheric modification, alter conidial quality of Beauveria bassiana.

AIM: The aim of this study was to determine the relationship between reactive oxygen species (ROS) production and conidial infectivity in Beauveria bassiana. METHODS AND RESULTS: Beauveria bassiana Bb 882.5 was cultured in solid-state culture (SSC) using rice under three oxygen conditions (21%, or pulses at 16 and 26%). Hydrophobicity was determined using exclusion phase assay. Bioassays with larvae or adults of Tenebrio molitor allowed the measurements of infectivity parameters. A fluorometric method was used for ROS quantification (superoxide and total peroxides). NADPH oxidase (NOX) activity was determined by specific inhibition. Conidial hydrophobicity decreased by O2 pulses. Mortality of larvae was only achieved with conidia harvested from cultures under 21% O2 ; whereas for adult insects, the infectivity parameters deteriorated in conidia obtained after pulses at 16 and 26% O2 . At day 7, ROS production increased after 16 and 26% O2 treatments. NOX activity induced ROS production at early stages of the culture. CONCLUSION: Modification of atmospheric oxygen increases ROS production, reducing conidial quality and infectivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in which conidial infectivity and ROS production in B. bassiana has been related, enhancing the knowledge of the effect of O2 pulses in B. bassiana.

Production of conidia of Beauveria bassiana in solid-state culture: current status and future perspectives.

Beauveria bassiana is an important entomopathogenic fungus widely commercialized in the world. Recent progress and achievements on conidia production have focused on a yield goal of 10(9) to 10(10) conidia per gram of dry substrate. Due to cost-competitive perspectives, these yields should be associated with better production rates or productivities. This study presents a review of relevant studies of B. bassiana conidia production on solid-state cultures and the parameters that should be taken into account to maintain constant quality in the product to be commercialized. Conditions for maximizing production and infectivity of B. bassiana conidia are also analysed.

Detoxification and mineralization of Acid Blue 74: study of an alternative secondary treatment to improve the enzymatic decolourization.

Many reports describe the decolourization of dyes by fungal enzymes. However, these enzymes do not contribute to dye mineralization but only to its biotransformation into less coloured or colourless molecules persisting in solution. Therefore, it is essential to analyse the identity of the metabolites produced during enzymatic treatments and its biodegradation into an appropriate system. The present work examines the decolourization/detoxification of a simulated effluent (containing Acid Blue 74) by fungal enzymes and proposes a secondary treatment using an anaerobic system to improve the enzymatic decolourization through the complete mineralization of the dye. Ligninolytic enzymes were produced by solid culture using the thermo-tolerant fungus Fomes sp. EUM1. The enzymes produced showed a high rate of decolourization (>95 % in 5 h) and were stable at elevated temperature (40 °C) and ionic strength (NaCl, 50 mM). Isatin-5-sulphonic acid was identified via (1)H-NMR as oxidation product; tests using Daphnia magna revealed the non-toxic nature of this compound. To improve the enzymatic degradation and avoid coupling reactions between the oxidation products, the effluent was subjected to an anaerobic (methanogenic) treatment, which achieved high mineralization efficiencies (>85 %). To confirm the mineralization of isatin-5-sulphonic acid, a specific degradation study, which has not been reported before, with this single compound was conducted under the same conditions; the results showed high removal efficiencies (86 %) with methane production as evidence of mineralization. These results showed the applicability of an anaerobic methanogenic system to improve the enzymatic decolourization/detoxification of Acid Blue 74 and achieve its complete mineralization.

Homologue expression of a ß-xylosidase from native Aspergillus niger.

Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. ß-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-ß-xylanase activity. This work reports the partial characterization of a purified ß-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding ß-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. ß-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein(-1) at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol(-1), and at 50°C optimum stability was shown at pH 4.0-5.0. ß-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), ß-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn(2+). Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.

Selection of improved Beauveria bassiana (Bals.) Vuill. strains based on 2-deoxy-D-glucose resistance and physiological analysis.

A series of 2-deoxy-D-glucose resistant mutants was obtained from wild type Beauveria bassiana 88 (Bb 88) by UV irradiation. Five mutants were characterized on Sabouraud Dextrose Agar and Chitin Agar for both radial extension rate (V(r)) and specific growth rate (micro). These values were obtained after adjusting morphometric data to a mathematical model used for filamentous fungi. Additionally, the protease and lipase potency index, conidial size, viability, and production levels were analyzed. The highest values for those physiological measurements were obtained by mutant 882.5 which, relative to Bb 88, showed a 30% reduction in half-life (LT(50)) on Sphenarium purpurascens, 70% on Acheta domesticus, and 71% on Tenebrio molitor larvae and adults. The half lethal concentration (LC(50)) on T. molitor larvae was 2.8 x 10(5)conidia/mL (con/mL) and 1.5 x 10(6)con/mL, respectively, for mutant 882.5 and Bb 88. This demonstrates that mutant 882.5 is more virulent, with up to an 80% reduction in LC(50). This work provides a convenient method for improving strains to be used in biocontrol as a suitable alternative to transgenic constructs.

Comparison of two estrus-synchronization protocols and timed artificial insemination in dairy cattle.

The objective of this study was to evaluate the effect of the Ovsynch protocol with and without exogenous progesterone on pregnancy rate (PR) in cows in which estrous cycles were previously synchronized with 2 doses of PGF(2alpha) and that were not detected in estrus during the presynchronization period. The study was conducted in Chihuahua, Mexico (8,650 Holstein milking cows; 305-d mature equivalent milk yield = 13,790 kg). On d 47 postpartum, estrous cycles in cows were synchronized by using 2 doses of PGF(2alpha) 14 d apart. Any cow detected in estrus during this presynchronization period was inseminated. Cows not detected in estrus were selected at random and assigned to receive progesterone supplementation or to serve as controls. Controls (n = 594) were subjected to the Ovsynch protocol and cows in the progesterone supplemented treatment (n = 594) were subjected to the Ovsynch protocol plus an intravaginal insert containing 1.9 g of progesterone inserted at the time of the first GnRH injection and removed 7 d later. Progesterone-supplemented cows had a greater PR (31.2%) compared with controls (22.7%). Plasma progesterone concentrations at artificial insemination (AI) were <1 ng/mL and did not differ between treatments. At 14 d post-AI, however, more cows that received progesterone supplementation had concentrations of progesterone >1 ng/mL compared with controls. It was concluded that after a presynchronization period, cows subjected to the Ovsynch program and supplemented with exogenous progesterone had a greater PR and greater concentrations of progesterone after AI than those subjected to the Ovsynch protocol and not supplemented with progesterone.

Growth of Pleurotus ostreatus on wheat straw and wheat-grain-based media: Biochemical aspects and preparation of mushroom inoculum.

Mycelial growth, intracellular activity of proteases, laccases and beta-1,3-glucanases, and cytoplasmic protein were evaluated in the vegetative phase of Pleurotus ostreatus grown on wheat straw and in wheat-grain-based media in Petri dishes and in bottles. The productivity of the wheat straw and wheat-grain-based spawn in cylindrical polyethylene bags containing 5 kg of chopped straw was also determined. We observed high activity of proteases and high content of intracellular protein in cultures grown on wheat straw. This suggests that the proteases are not secreted into the medium and that the protein is an important cellular reserve. On the contrary, cultures grown on wheat straw secreted laccases into the medium, which could be induced by this substrate. P. ostreatus grown on media prepared with a combination of wheat straw and wheat grain showed a high radial growth rate in Petri dishes and a high level of mycelial growth in bottles. The productivities of wheat straw and wheat-grain-based spawn were similar. Our results show that cheaper and more productive mushroom spawn can be prepared by developing the mycelium on wheat straw and wheat-grain-based substrates.

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 (r(r)) 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.

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.