Toxicity of Bacillus thuringiensis var. israelensis in aqueous suspension on the South American common frog Leptodactylus latrans (Anura: Leptodactylidae) tadpoles.

The effects of commercial formulations of Bacillus thuringiensisvar.israelensis (Bti) on non-target organisms are still a matter of debate; in amphibians, the risks of Bti are little known. To evaluate the toxicity of a commercial liquid (aqueous suspension, AS) formulation of Bti (Introban(®)) on Leptodactylus latrans tadpoles, including median lethal concentration (LC50) and no-and lowest-observed-effect concentrations (NOEC and LOEC, respectively), as well as the possible effects of Bti on oxidative responses, erythrocytes genotoxicity, and histology of the intestines. In the laboratory, tadpoles were exposed to nominal concentrations of 0 (control), 2.5, 5, 10, 20 and 40 mg/L of formulated Bti-AS. Glutathione S-transferase (GST) and catalase (CAT) activities, as well as formation of erythrocyte nuclear abnormalities (ENAs), and histological effect were measured in tadpoles displaying survival rates >85%. L. latrans tadpoles were sensitive to exposure to Bti-AS, reaching 100% mortality after 48 h of exposure at the highest concentration. Bti-AS induced GST and CAT enzymes and genotoxicity (erythrocyte's nuclear abnormalities), and caused intestine's histopathology. Our results demonstrate that toxicity of Bti-AS is dose-dependent for L. latrans tadpoles and that sublethal exposure alters enzymes of oxidative stress, induces genotoxicity, and causes intestine damage. Further research is needed to evaluate the ecotoxicological risk of the massive use of Bti formulations on amphibian populations that commonly used suburban wastewater or urban waterbodies to reproduce and where this biopesticide is frequently applied.

Rational design of a culture medium for the intensification of lipid storage in Chlorella sp. Performance evaluation in air-lift bioreactor.

An optimal medium to culture Chlorella sp., microalgae capable of storage intracellular lipids was obtained. This culture medium consists of a saline base plus carbon-energy and nitrogen sources. Significant factors exerting influence on the culture parameters were selected. Then, by applying response surface methodology coupled to desirability function, an optimal formulation, specific for the heterotrophic growth of Chlorella sp. that allows maximizing lipid concentration was obtained. During the experimental verification, the possibility of replacing commercial glucose by hydrolysates obtained from lignocellulosic materials was evaluated. Biochemical hydrolysate of corn bran allowed obtaining important improvements in lipid concentration. Finally, the optimal formulation was evaluated in an air-lift bioreactor performing a fed-batch culture. Culturing the strain in these conditions allowed rising lipid concentrations.

Significant factors selection in the chemical and enzymatic hydrolysis of lignocellulosic residues by a genetic algorithm analysis and comparison with the standard Plackett-Burman methodology.

A comparison between the classic Plackett-Burman design (PB) ANOVA analysis and a genetic algorithm (GA) approach to identify significant factors have been carried out. This comparison was made by applying both analyses to data obtained from the experimental results when optimizing both chemical and enzymatic hydrolysis of three lignocellulosic feedstocks (corn and wheat bran, and pine sawdust) by a PB experimental design. Depending on the kind of biomass and the hydrolysis being considered, different results were obtained. Interestingly, some interactions were found to be significant by the GA approach and allowed to identify significant factors, that otherwise, based only in the classic PB analysis, would have not been taken into account in a further optimization step. Improvements in the fitting of c.a. 80% were obtained when comparing the coefficient of determination (R2) computed for both methods.

Application of response surface methodology and artificial neural networks for optimization of recombinant Oryza sativa non-symbiotic hemoglobin 1 production by Escherichia coli in medium containing byproduct glycerol.

Production of recombinant Oryza sativa non-symbiotic hemoglobin 1 (OsHb1) by Escherichia coli was maximized in shake-flask cultures in media containing tryptone, yeast extract, sodium chloride and byproduct glycerol from biodiesel production. Response surface methodology (RSM) and artificial neural networks (ANNs), followed by multiple response optimization through a desirability function were applied to evaluate the amount of OsHb1 produced. The results obtained by the application of ANNs were more reliable since better statistical parameters were obtained. The optimal conditions were (gL(-1)), tryptone, 42.69; yeast extract, 20.11; sodium chloride, 17.77; and byproduct glycerol, 0.33. A maximum recombinant protein concentration of 3.50gL(-1) and a minimum biomass concentration of 18.48gL(-1) were obtained under these conditions. Although the concentrations of tryptone, yeast extract and sodium chloride are relatively high, the increase in the yield with respect to biomass formed (Y(P/X)) overcomes this disadvantage.

Application of response surface methodology and artificial neural networks for optimization of recombinant Oryza sativa non-symbiotic hemoglobin 1 production by Escherichia coli in medium containing byproduct glycerol.

Production of recombinant Oryza sativa non-symbiotic hemoglobin 1 (OsHb1) by Escherichia coli was maximized in shake-flask cultures in media containing tryptone, yeast extract, sodium chloride and byproduct glycerol from biodiesel production. Response surface methodology (RSM) and artificial neural networks (ANNs), followed by multiple response optimization through a desirability function were applied to evaluate the amount of OsHb1 produced. The results obtained by the application of ANNs were more reliable since better statistical parameters were obtained. The optimal conditions were (g L(-1)), tryptone, 42.69; yeast extract, 20.11; sodium chloride, 17.77; and byproduct glycerol, 0.33. A maximum recombinant protein concentration of 3.50 g L(-1) and a minimum biomass concentration of 18.48 g L(-1) were obtained under these conditions. Although the concentrations of tryptone, yeast extract and sodium chloride are relatively high, the increase in the yield with respect to biomass formed (Y(P/X)) overcomes this disadvantage.

Purification and characterization of a glutathione reductase from Phaeodactylum tricornutum.

Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14 microM and 60 microM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.