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.

Influence of an aerobic fungus grown on solid culture on ruminal degradability and on a mixture culture of anaerobic cellulolytic bacteria.

In this work, the effect of a solid fungal culture of Aspergillus niger (An) grown on coffee pulp on the in situ ruminal degradability (RD) of corn stover was evaluated. In addition, the effect of its extracts on the in vitro dry matter disappearance (IVDMD) and on a mixed culture of anaerobic cellulolytic bacteria (MCACB) was also investigated. The solid ferment was a crude culture of An, grown on coffee pulp. Regarding in situ RD, a significant difference (p < 0.05) was found between treatment with 200 g/day of the solid culture and control (no solid culture added) on dry matter, crude protein and neutral detergent fibre on RD. All the water extracts (pH 4, 7 and 10) enhanced IVDMD and stimulated the cellulolytic activity on a MCACB. Ultrafiltration results showed that active compounds with a molecular weight lower than 30 kDa were responsible for the effect on MCACB. Such results suggest that the effects of the solid An culture in RD are related to the presence of water soluble compounds having a molecular weight lower than 30 kDa.

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.

New approach for selecting pectinase producing mutants of Aspergillus niger well adapted to solid state fermentation.

The aim of this paper is to review and study a new approach for improving strains of Aspergillus niger specially adapted to produce pectinases by Solid State Fermentation (SSF) with materials having low levels of water activity (a(w)), i.e., coffee pulp. Special emphasis is placed on the use of two antimetabolic compounds: 2-deoxy-glucose (DG) and 2,4-dinitro-phenol (DNP) combined with a water depressant (ethylene glycol = EG) in order to put strong selection pressures on UV treated spores from parental strain C28B25 isolated from a coffee plantation. Such a strain was found to be DG sensitive. Results suggested the existence of a reciprocal relation between adaptation of isolated strains to SSF or to Submerged Fermentation (SmF) systems. Preliminary physiological analysis of isolated strains showed that at least some few initially DG resistant mutants could revert to DG sensitive phenotype but conserving increased pectinase production. Also it was found that phenotype for DNP resistance could be associated to changes of DG resistance. Finally, it was found that low levels of a(w) produced by adding 15% EG to agar plates, were a significant selection factor for strains well adapted to SSF system.

Overproduction of rifamycin B by Amycolatopsis mediterranei and its relationship with the toxic effect of barbital on growth.

A novel method for selecting overproducing strains of rifamycin B was developed. This technique involves the use of lysozyme and the effect of barbital on the growth of A. mediterranei. Complete medium added with glycine and barbital was inoculated with mutagenized mycelium, incubated for 48 hours and treated with lysozyme. The lysozyme resistant mycelium was washed with dilute detergent. Complete medium with glycine but without barbital was inoculated with the washed mycelium. Protoplasts were obtained and regenerated and the colonies were picked and seeded on Bennet agar plates with and without barbital. Two selected mutants were sensitive to 0.5% barbital producing 200% more rifamycin than the parental strain. In addition, 30 barbital resistant mutants were isolated and their production level was lower than the one observed with the parental strain. These results suggest that the effect of barbital on secondary metabolism (rifamycin production) is related to its effect on primary metabolism.

Denitrification in presence of benzene, toluene, and m-xylene: kinetics, mass balance, and yields.

Denitrification of the electron donors toluene-C (15-100 mg/L), m-xylene-C (15-70 mg/L), benzene-C (5-25 mg/L), and acetate-C as experimental reference (50-140 mg/L) was carried out in batch culture. An initial concentration of 1.1 +/- 0.15 g of volatile suspended solids/L of denitrifying sludge without previous exposure to aromatic compounds was used as inoculum. The results showed toluene and nitrate consumption efficiency (ET and EN, respectively) of 100%. Toluene was completely mineralized (oxidized) to CO2. In all cases, the N2 (YN2) and HCO3-yields (YHCO3) were 0.97 +/- 0.01 and 0.8 +/- 0.05, respectively. The consumption efficiency (EX) of m-xylene (53 +/- 5.7%) was partial. The YN2 and YHCO3 were 0.96 +/- 0.01 and 0.86 +/- 0.02, respectively. Benzene was not consumed under denitrifying conditions. The specific consumption rates of toluene (qT) and m-xylene (qX) were lower than that of acetate (qA). The differences in specific consumption rates were probably owing to the negative effect of benzene, toluene, and isomers of xylene on the cell membrane.

Thermostability of native and pegylated Myceliophthora thermophila laccase in aqueous and mixed solvents.

A commercial preparation of laccase (EC 1.10.3.2), cloned from Myceliophthora thermophila and expressed in Aspergillus oryzae (MtL), was purified and modified by conjugation with poly(ethylene glycol) (M(r) = 5000) and is labeled PEG-MtL. Native enzyme was found to have a molecular mass of 80 kDa, as determined by gel filtration, and 110 kDa, by SDS-PAGE. The oxidative dimerization of 2,6-dimethoxyphenol (DMP) to produce the corresponding dibenzoquinone was catalyzed by MtL in a manner comparable to that for a diffusion-controlled reaction (k(cat)/K(M) approximately = 10(8) M(-)(1) s(-)(1) and E(a) approximately = 18 kJ M(-)(1)). PEG-MtL was found, by TNBS titration, to have blocked 54% of lysine groups; its hydrodynamic and charge properties were different from those of MtL. Catalytic efficiency (k(cat)/K(M)) of PEG-MtL was similar to that of MtL with DMP as substrate; however, k(cat)/K(M) was 2-fold reduced for the reaction in which 2',2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is oxidized to form a radical cation. E(a) values were similar in both enzyme preparations when assayed in buffered solutions. Far-UV CD spectra were similar for MtL and PEG-MtL and consistent with a protein rich in beta-sheet structure with negligible content of alpha-helices. A blue shift of near-UV CD spectrum for PEG-MtL as compared to MtL was consistent with the decreased polarity of the tyrosyl side chains upon PEG conjugation. Also the blue band of the copper active site was shifted from lambda approximately 610 nm (MtL) to lambda approximately 575 nm (PEG-MtL). Scanning microcalorimetry showed small denaturation enthalpies (6.3 and 7.5 J g(-)(1) for MtL and PEG-MtL, respectively), indicating the high stability of the beta-sheet folding pattern of laccases. However, PEG-MtL proved to be more stable, its half-denaturation temperature being 2 degrees C higher than that of MtL. In 30% alcohol, pegylated laccase showed slower enzyme-activity decay rates than the unmodified enzyme; this behavior was caused by a decrease in the activation entropy of the denaturation reaction. Results can be explained by entropic stabilization by PEG conjugation because of the restricted motion of some surface amino acid side chains, which results in a more stable active site.

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study.

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A(w)=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A(w) in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination.

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid.

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF.

Heat transfer simulation in solid substrate fermentation.

A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.

Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation.

Three extracellular pectinases were produced by Aspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (Km values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but the Km values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number of protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced by A. niger.

Morphometric evaluation of the specific growth rate of Aspergillus niger grown in agar plates at high glucose levels.

Development of surface grown cultures of Aspergillus niger no. 10 was studied at two experimental levels: (a) following the time course of the biomass density (X [=] mg cm(-2)) and fitting the data by the logistic expression, which yielded a macroscopic specific growth rate expressed as micro(obs) = (dX/Xdt)[1-(X/X(max))](-1); and (b) measuring morphometric parameters like the specific elongation rate (k) of the germ tubes and their diameters (D(h)), the colony rate of radial extension (u(r)), and the mean length of distal hyphae (L(av)) to estimate the specific growth rate with the following proposed expression: micro(calc) = u(r)ln2[L(av)ln(L(av)/D(h))](-1). Increases in the initial glucose concentration (10, 40, 70, 120, 200, and 300 g L(-1)) caused reductions in the specific growth rates, the elongation kinetics of the germ tubes, and the hyphal diameter, nevertheless, u(r) and X(max) presented parabolic behavior, showing their maxima in the interval of 90 to 120 g L(-1) of glucose. The overall macroscopic effect of the tested concentrations of glucose on surface grown cultures of A. niger was to produce densely packed and slowly extending colonies, where changes in hyphal lengths and diameters were significant. There was good agreement between micro(obs) and micro(calc) values. Hence, this work validates a kinetic model based on morphometric data to estimate the specific growth rate of molds, obtained from dry weight data, using mold cultures grown in the same solid medium i.e., agar plates.

Symmetric branching model for the kinetics of mycelial growth.

A mathematical model, linking microscopic to macroscopic parameters of the kinetics of mycelial growth is presented. The model consists of two parts: (a) a microscopic description, based on the assumption that growth of a mycelium can be represented approximately by the growth of a symmetric binary tree, where the branching level (microscopic state variable) is logarithmically related to the number of tips and segments; and (b) a macroscopic description which makes use of the microscopic description in order to define the parameters related to the evolution of biomass (macroscopic state variable) as a function of time. The latter uses a distribution of arrested tips in a population of mycelia, in order to estimate the fraction of non-growing biomass in terms of a power law function with coefficient, n, of the biomass concentration. The microscopic description explains the fact that the germ tube specific growth rate of Aspergillus nidulans measured in a growth chamber, is about the double the specific growth rate of this organism, when measured in shake flasks. It predicts that the length of the hyphal growth unit of the mycelium of Geotrichum candidum would be approximately the double the germ tube length measured at the time just before the first branching event. It also allows the derivation of useful expressions for predicting macroscopic parameters, such as the maximal specific growth rate, the initial amount of biomass, and the amount of biomass before the branching process starts. Those estimates are done in terms of microscopic quantities, i.e., the amount of germinated spores, the diameters of the spores and hyphae, the average rate of tip extension, and the average internodal segment length. Estimation of coefficient n by fitting the macroscopic description to a growth curve of A. niger gives an indication on the degree of skewness of the distribution of arrested mycelia. Estimated macroscopic parameters are in relative good agreement with measured average segment length.

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations.

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.