Pioneer access of the foam nest bacterial community of Leptodactylidae frogs and its biotechnological potential.

Many anuran amphibians deposit their eggs in foam nests, biostructures that help protect the eggs and tadpoles from predators. Currently, there are no other identification and description studies of the cultivable microbiota role in the nests of the Leptodactylid frogs such as Physalaemus cuvieri, Leptodactylus vastus and Adenomera hylaedactyla. This study aimed to isolate and identify the culturable bacteria from these three anuran species' nests, as well as to prospect enzymes produced by this microbiota. Foam nests samples and environmental samples were diluted and viable cell count was determined. Bacterial morphotypes from foam nest samples were isolated through spread plate technique. Isolates' DNAs were extracted followed by rRNA 16S gene amplification and Sanger sequencing. To evaluate their enzymatic potential, the isolates were cultured in ATGE medium supplemented with starch (0.1% w/v), gelatin (3% w/v) and skimmed milk (1% w/v), to verify amylase and protease activity. A total of 183 bacterial morphotypes were isolated, comprising 33 bacterial genera. Proteobacteria phylum was the most abundant in all the three nests (79%). The genera Pseudomonas and Aeromonas were the most abundant taxon in P. cuvieri and L. vastus. In A. Hylaedactyla, were Enterobacter and Bacillus. Regarding enzymatic activities, 130 isolates displayed protease activity and 45 isolates were positive for amylase activity. Our results provide unprecedented information concerning culturable bacterial microbiota of the foam nests of the Leptodactylid frogs, as well as their potential for biomolecules of biotechnological interest.

Sustainable recovery of protein-rich liquor from shrimp farming waste by lactic acid fermentation for application in tilapia feed.

The biotransformation of shrimp head waste into ingredients for Nile tilapia (Oreochromis niloticus) post-larvae (PLs) diet formulations was evaluated herein. A novel consortium of lactic acid bacteria, comprising the strains Lactobacillus futsaii LAB06 and L. plantarum LAB14, selected based on kinetic growth parameters, was applied in the fermentation of shrimp head waste. After 48 h, the highest lactic acid production was 100 g L-1, allowing for maximum recovery of chitin and protein-rich liquor. The liquor was added to commercial powdered fish feed at 15, 30, and 45% (w/w) and offered to Nile tilapia PL during 28 days. The inclusion of 30% provided the best results for Nile tilapia PL survival, weight and length gains, specific growth rate, and biomass, proving that the fermented liquor can be incorporated, leading to economic benefits and contributing to the reduction of environmental pollution caused by the improper disposal of shrimp waste.

Bioaugmentation strategy employing a microbial consortium immobilized in chitosan beads for oil degradation in mesocosm scale.

A bacterial consortium composed by four metagenomic clones and Bacillus subtilis strain CBMAI 707, all derived from petroleum reservoirs, was entrapped in chitosan beads and evaluated regarding hydrocarbon degradation capability. Experiments were carried out in mesocosm scale (3000L) with seawater artificially polluted with crude oil. At different time intervals, mesocosms were sampled and subjected to GC-FID and microbiological analyses, as total and heterotrophic culturable bacterial abundance (DAPI and CFU count), biological oxygen demand (BOD) and taxonomic diversity (massive sequencing of 16S rRNA genes). The results obtained showed that degradation of n-alkane hydrocarbons was similar between both treatments. However, aromatic compound degradation was more efficient in bioaugmentation treatment, with biodegradation percentages reaching up to 99% in 30days. Community dynamics was different between treatments and the consortium used in the bioaugmentation treatment contributed to a significant increase in aromatic hydrocarbon degradation.

A yeast isolated from cashew apple juice and its ability to produce first- and second-generation ethanol.

The aim of this study was to isolate and identify an indigenous yeast from cashew apple juice (CAJ) and then use it in the production of first- and second-generation ethanol, using CAJ and the enzymatic hydrolysate of cashew apple bagasse (MCAB-OH), respectively. The isolated yeast was identified as belonging to the genus Hanseniaspora. Afterward, the effect of the medium initial pH on the production of ethanol from CAJ was evaluated in the range of 3.0 to 5.5, with its maximum ethanol production of 42 g L(-1) and Y P/S of 0.44 g g(-1) and 96 % efficiency. The effect of temperature (28-38 °C) on ethanol production was evaluated in a synthetic medium, and no difference in ethanol production in the temperature range evaluated (28-36 °C) was observed. At 32 °C, the yield, concentration, efficiency, and productivity of ethanol when using the CAJ medium were higher when compared to the results achieved for the synthetic medium. Regarding second-generation ethanol, the results showed that the yeast produced 24.37 g L(-1) of ethanol with an efficiency of 80.23 % and a productivity of 4.87 g L(-1) h(-1) at 5 h. Therefore, Hanseniaspora sp., isolated from CAJ, is a promising microorganism for the production of first- and second-generation ethanol.

Screening of biosurfactant-producing Bacillus strains using glycerol from the biodiesel synthesis as main carbon source.

Glycerol, a co-product of biodiesel production, was evaluated as carbon source for biosurfactant production. For this reason, seven non-pathogenic biosurfactant-producing Bacillus strains, isolated from the tank of chlorination at the Wastewater Treatment Plant at Federal University of Ceara, were screened. The production of biosurfactant was verified by determining the surface tension value, as well as the emulsifying capacity of the free-cell broth against soy oil, kerosene and N-hexadecane. Best results were achieved when using LAMI005 and LAMI009 strains, whose biosurfactant reduced the surface tension of the broth to 28.8 ± 0.0 and 27.1 ± 0.1 mN m(-1), respectively. Additionally, at 72 h of cultivation, 441.06 and 267.56 mg L(-1) of surfactin were produced by LAMI005 and LAMI009, respectively. The biosurfactants were capable of forming stable emulsions with various hydrocarbons, such as soy oil and kerosene. Analyses carried out with high performance liquid chromatography (HPLC) showed that the biosurfactant produced by Bacillus subtilis LAMI009 and LAMI005 was compatible with the commercially available surfactin standard. The values of minimum surface tension and the CMC of the produced biosurfactant indicated that it is feasible to produce biosurfactants from a residual and renewable and low-cost carbon source, such as glycerol.

New approach for petroleum hydrocarbon degradation using bacterial spores entrapped in chitosan beads.

Spores of Bacillus subtilis LAMI008 were entrapped in 3-mm chitosan beads and cross-linked with 0.3% glutaraldehyde for n-hexadecane biodegradation and biosurfactant recovery. When exposed to nutrients, the spores generated vegetative cells without morphological alterations as revealed by atomic force microscopy. The entrapped cells degraded almost 100% of 1% of n-hexadecane in medium supplemented with 1% glucose and produce biosurfactant within 48 h, as well as free cells. The number of viable cells inside the beads was maintained throughout the n-hexadecane degradation process and the released biosurfactant was not used as a carbon source. Entrapment of bacterial spores in chitosan beads overcomes problems with stability, storage, and long term cell viability encountered with vegetative cells. This approach can potentially be utilized for biodegradation of complex compounds by entrapping spores of different species of bacteria.

SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth.

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD(50) 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1cm)(1%) of 6.3, and fluorescence emission in the 290-450 nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 microg/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens.

Immunological and allergenic responses induced by latex fractions of Calotropis procera (Ait.) R.Br.

Immunological and allergenic responses against the latex of Calotropis procera were investigated in mice by oral and subcutaneous routes. The latex was fractionated according to water solubility and molecular size of its components. The fractions were named as non-dialyzable latex (NDL) corresponding to the major latex proteins, dialyzable latex (DL) corresponding to low molecular size substances and rubber latex (RL) which was highly insoluble in water. Anti-sera against these fractions were assayed for total IgG and IgA titration by ELISA and IgE and IgG(1) were quantified by passive cutaneous anaphylaxis (PCA) in rats and mice, respectively. None of the fractions induced antibodies level increases when mice received latex fractions by oral route and thus, did not develop allergy. Nonetheless, anti-sera of mice sensitized with NDL and RL by subcutaneous route displayed considerable immunological response while DL did not. IgG level augmented consistently against NDL and RL while IgA response was detected only to NDL. NDL and RL induced very strong PCA reactions suggesting that both fractions would contain latex substances involved in allergy. Furthermore, protein analysis of NDL and RL suggests that RL still retain residual proteins abundantly found in NDL that could explain its similar allergenic effect. No IgG(1) reaction was detected in any of the anti-sera tested. According to the results, the proteins of latex of Calotropis procera can provoke allergy by subcutaneous route. The NDL has previously shown to display anti-inflammatory and analgesic activities by intraperitoneal injection. It should be relevant to determine whether NDL could induce such activities when assayed by oral route since it was ineffective to induce allergy by this way.

Differential activity of a lectin from Solieria filiformis against human pathogenic bacteria

A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium). The lectin (500 æg/mL) stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 æg/mL but the lectin (10-1000 æg/mL) had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline) were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 æg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 æg/mL), its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.

Differential activity of a lectin from Solieria filiformis against human pathogenic bacteria.

A lectin isolated from the red alga Solieria filiformis was evaluated for its effect on the growth of 8 gram-negative and 3 gram-positive bacteria cultivated in liquid medium (three independent experiments/bacterium). The lectin (500 microg/mL) stimulated the growth of the gram-positive species Bacillus cereus and inhibited the growth of the gram-negative species Serratia marcescens, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus sp, and Pseudomonas aeruginosa at 1000 microg/mL but the lectin (10-1000 microg/mL) had no effect on the growth of the gram-positive bacteria Staphylococcus aureus and B. subtilis, or on the gram-negative bacteria Escherichia coli and Salmonella typhimurium. The purified lectin significantly reduced the cell density of gram-negative bacteria, although no changes in growth phases (log, exponential and of decline) were observed. It is possible that the interaction of S. filiformis lectin with the cell surface receptors of gram-negative bacteria promotes alterations in the flow of nutrients, which would explain the bacteriostatic effect. Growth stimulation of the gram-positive bacterium B. cereus was more marked in the presence of the lectin at a concentration of 1000 microg/mL. The stimulation of the growth of B. cereus was not observed when the lectin was previously incubated with mannan (125 microg/mL), its hapten. Thus, we suggest the involvement of the binding site of the lectin in this effect. The present study reports the first data on the inhibition and stimulation of pathogenic bacterial cells by marine alga lectins.

Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of antinutritional and toxic proteins.

The research was conducted with two different recently released Brazilian soybean cultivars (Rio Balsas and Bays) to evaluate whether there is any correlation between the different levels of antinutritional and/or toxic proteins in the cultivars and their nutritive value as sources of protein for monogastric animals (rats). Furthermore, it is discussed, for the first time, the role of the dietary soyatoxin on the performance of rats fed on diets containing soyatoxin-rich (cv. Bays) and soyatoxin-free (cv. Rio Balsas) soybean cultivars. Feeding rats with diets containing raw soybean cultivars showed a lower growth rate, net protein utilization and digestibility, a much higher dry matter and nitrogen excretion and macroscopic alterations in internal organs when compared to rats fed on egg-white protein. The nutritional parameters measured for the diet based on raw Bays cultivar were poorer than those of the diet prepared with Rio Balsas. In the raw soybeans, trypsin inhibitor and lectin, and urease to a lesser extent, significantly affected at different fashion the soybean protein utilization. Heating treatment of the Bays seeds increased the growth rate, NPU, in vivo protein digestibility and practically eliminated or attenuated all the organ alterations observed. This study might be helpful in the choice of safe and nutritious soybean cultivars.

Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, 1828

The antimicrobial, hemagglutinating and toxic activities of the purple fluid of the sea hare Aplysia dactylomela are described. Intact or dialyzed purple fluid inhibited the growth of species of Gram-positive and Gram-negative bacteria and the action was not bactericidal but bacteriostatic. The active factor or factors were heat labile and sensitive to extreme pH values. The fluid preferentially agglutinated rabbit erythrocytes and, to a lesser extent, human blood cells, and this activity was inhibited by the glycoprotein fetuin, a fact suggesting the presence of a lectin. The fluid was also toxic to brine shrimp nauplii (LD 50 141.25 µg protein/ml) and to mice injected intraperitoneally (LD 50 201.8 ñ 8.6 mg protein/kg), in a dose-dependent fashion. These toxic activities were abolished when the fluid was heated. Taken together, the data suggest that the activities of the purple fluid are due primarily to substance(s) of a protein nature which may be involved in the chemical defense mechanism of this sea hare