Antioxidant compounds extracted from Diaporthe schini using supercritical CO2 plus cosolvent.

Endophytic fungi have been highlight in the production of secondary metabolites with different bioactive properties, such as in the production of the antioxidant compounds. Therefore, the objective of this work was the extraction of the antioxidant compounds from the biomass of Diaporthe schini using supercritical carbon dioxide (CO2) without and with ethanol as cosolvent. The biomass was produced by submerged fermentation and the parameters evaluated in the extraction process were: pressure (150-250 bar), temperature (40-60 ºC) and cosolvent [biomass: cosolvent ratio, 1:0, 1:0.75 and 1:1.5 (w/v)]. Extraction yield, antioxidant activity and chemical composition of the extracts were determined. The highest extraction yield (3.24 wt.%) and the best antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (96.62%) were obtained at 40 ºC, 250 bar and biomass:cosolvent ratio of 1:1.5 (w/v). The chemical compounds 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0]nonane and benzeneethanol identified in GC/MS could be responsible for the antioxidant activity found in this study.

Separation of microbial oil produced by Mortierella isabellina using polymeric membranes.

The objective of this work was to concentrate, through a membrane separation process, the fatty acids from oil/solvent mixture. The oil was obtained by ultrasound-assisted extraction from freeze-dried cells of Mortierella isabellina. The concentration of the fatty acids was investigated using flat-sheet polymer membranes of ultrafiltration and nanofiltration. The effects of temperature and pressure were evaluated by the retention of the fatty acids. Oil retentions between 45.23 and 58.20% to ultrafiltration membrane and 43.50 and 56.00% to nanofiltration membrane were observed. The best condition for the ultrafiltration membrane was 4 bar and 40 °C and for nanofiltration membrane was 12 bar and 50 °C. The oil contains a high concentration of oleic acid and palmitic acid that is a desirable property for the biodiesel production. The results showed the applicability of this technology in the solvent recovery step whereas the oil recovered contains a high concentration of fatty acids.

Production of metabolites with antioxidant activity by Botryosphaeria dothidea in submerged fermentation.

Antioxidants are able of inhibiting free radicals and play an important role in human diet, protection of foods, anti-aging cosmetics, among others. Fungi have been highlighted in production of metabolites with biological activity, such as antioxidant. The main objective of this study was to select a fungus from resources of Brazilians Pampa and Atlantic Forest biomes with hability for production of antioxidants by submerged fermentation. The scavenging activity of the compounds was determined against radical 2.2-diphenyl-1-picrylhydrazyl (DPPH), superoxide and hydroxyl. Botryosphaeria dothidea exhibited higher potential for the production of antioxidants, and its supernatant showed percentage inhibition values of 94.47, 94.87 and 89.78% against DPPH, superoxide and hydroxyl radicals and IC50 of 0.206 mg/mL. The identification of the volatile compounds present in the broth verified the presence of the hexahydropyrrolizin-3-one; 1.2-benzenedicarboxylic acid bis(2-methylpropyl) ester and 3.6-bis(2-methylpropyl)-2.5-piperazinedione. According to this study, Botryosphaeria dothidea presents great potential to produce compounds with antioxidant activity.

Production of cutinase by solid-state fermentation and its use as adjuvant in bioherbicide formulation.

In the present study, it was presented a strategy to maximize the cutinase production by solid-state fermentation from different microorganisms and substrates. The best results were observed using Fusarium verticillioides, rice bran being the main substrate. Maximum yield of cutinase obtained by the strain was 16.22 U/g. For concentration, ethanol precipitation was used, and the purification factor was 2.4. The optimum temperature and pH for enzyme activity were 35 °C and 6.5, respectively. The enzyme was stable at a wide range of temperature and at all pH values tested. The concentrated cutinase was used as an adjuvant in a formulation containing cutinase + bioherbicide. The use of enzyme increased the efficiency of bioherbicide, since cutinase was responsible to remove/degrade the cutin that recovery the weed leaves and difficult the bioherbicide absorption. Cutinase showed to be a promising product to be used in formulation of bioherbicides.

Biosorption of crystal violet dye using inactive biomass of the fungus Diaporthe schini.

An inactive biomass of a new fungus recently discovered, Diaporthe schini, was evaluated for the biosorption of crystal violet (CV) in simulated textile effluents. The characterization assays were performed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and N2 adsorption/desorption isotherms. The influences of pH and biosorbent dosage on the biosorption capacity were evaluated. Kinetics, equilibrium and thermodynamic studies were also carried out. Characterization techniques showed an amorphous biosorbent, with a rough surface containing irregular particles and surface area of 6.5 m2 g-1. The most adequate values of pH and biosorbent dosage were 7.5 and 0.4 g L-1, respectively. The Elovich kinetic model and the Sips equilibrium model were suitable to fit the experimental data. The biosorption capacity increased with temperature, reaching a maximum biosorption capacity of 642.3 mg g-1 at 328 K. The biosorption was a spontaneous and endothermic process. Diaporthe schini inactive biomass was an interesting biosorbent to treat colored effluents, presenting efficiency of 87% in the decolorization of a simulated dye house effluent.

Degradation of Amaranth azo dye in water by heterogeneous photo-Fenton process using FeWO4 catalyst prepared by microwave irradiation.

FeWO4 particles were synthesized by a simple, rapid and facile microwave technique and their catalytic properties in heterogeneous photo-Fenton reaction were evaluated. This material was employed in the degradation of Amaranth azo dye. Individual and interactive effects of operational parameters such as pH, dye concentration and H2O2 dosage on the decolorization efficiency of Amaranth dye were evaluated by 2(3) central composite design. According to characterization techniques, a porous material and a well-crystallized phase of FeWO4 oxide were obtained. Regarding the photo-Fenton reaction assays, up to 97% color and 58% organic carbon removal were achieved in the best experimental conditions. In addition, the photo-Fenton process maintained treatment efficiency over five catalyst reuse cycles to indicate the durability of the FeWO4 catalyst. In summary, the results reveal that the synthesized FeWO4 material is a promising catalyst for wastewater treatment by heterogeneous photo-Fenton process.

Separation and purification of fructooligosaccharides on a zeolite fixed-bed column.

Fructooligosaccharides (FOS), a well-known prebiotic product, are obtained by enzymatic synthesis and consist of a mixture of mono- and disaccharides. In this work, a methodology for their separation and purification was developed using a zeolite fixed-bed column. The effects of column temperature (40-60°C), eluent flow rate (0.10-0.14 mL/min), injected to bed volume percent ratio (2.6-5.1%), and ethanol concentration in the eluent (40-60%, v/v) were investigated using a fractionary factorial design (2(4-1)), having the separation efficiency and purity as target responses. Additional experiments were performed as well, where the temperature and ethanol concentration were studied in a central composite design (2(2)). In this work, the zeolite fixed-bed column was shown to be a good alternative for FOS purification, allowing a FOS purity of 90% and separation efficiency of 6.86 between FOS and glucose, using an eluent at 45°C with 60% ethanol concentration.

Carbon nanotubes as supports for inulinase immobilization.

The commercial inulinase obtained from Aspergillus niger was non-covalently immobilized on multiwalled carbon nanotubes (MWNT-COOH). The immobilization conditions for the carbon nanotubes were defined by the central composite rotational design (CCRD). The effects of enzyme concentration (0.8%-1.7% v/v) and adsorbent:adsorbate ratio (1:460-1:175) on the enzyme immobilization were studied. The adsorbent:adsorbate ratio variable has positive effect and the enzyme concentration has a negative effect on the inulinase immobilization (U/g) response at the 90% significance level. These results show that the lower the enzyme concentration and the higher the adsorbent:adsorbate ratio, better is the immobilization. According to the results, it is possible to observe that the carbon nanotubes present an effective inulinase adsorption. Fast adsorption in about six minutes and a loading capacity of 51,047 U/g support using a 1.3% (v/v) inulinase concentration and a 1:460 adsorbent:adsorbate ratio was observed. The effects of temperature on the immobilized enzyme activity were evaluated, showing better activity at 50 °C. The immobilized enzyme maintained 100% of its activity during five weeks at room temperature. The immobilization strategy with MWNT-COOH was defined by the experimental design, showing that inulinase immobilization is a promising biotechnological application of carbon nanotubes.

Submerged cultivation of Nigrospora sp. in batch and fed-batch modes for microbial oil production.

Microbial lipids are a valuable source of potential biofuels and essential polyunsaturated fatty acids. The optimization of the fermentation conditions is a strategy that affects the total lipid concentration. The genus Nigrospora sp. has been the target of investigations based on its potential bioherbicidal action. Therefore, this study developed a strategy to maximize the biomass concentration and lipid accumulation by Nigrospora sp. in submerged fermentation. Different media compositions and process variables were investigated in shaken flasks and bioreactor in batch and fed-batch modes. Maximum biomass concentration and lipid accumulations were 40.17 g/L and 21.32 wt% in the bioreactor, which was 2.1 and 5.4 times higher than the same condition in shaken flasks, respectively. This study presents relevant information to the production of fungal lipids since few investigations are exploring the fed-batch strategy to increase the yield of fungi lipids, as well as few studies investigating Nigrospora sp. to produce lipids.

Evaluation of ultrasound waves for the production of chitinase and ß-1,3 glucanase by Trichoderma harzianum through SSF.

Cell wall degrading enzymes (chitinase and ß-1,3-glucanase) were produced by solid-state fermentation (SSF) using the fungus Trichoderma harzianum and different agro-industrial products, mainly residues. The influence of temperature (25-35 °C), initial moisture content (50-90% w/w), nutrient solution (1-2% v/w), and yeast extract (1-2% w/w) on enzyme activity was evaluated. The application of ultrasound during fermentation for different times (0-6 h/day) was also studied. White rice was the substrate that showed the highest chitinase and ß-1,3-glucanase activities, which were 31.31 U/g for chitinase and 23.83 U/g for ß-1,3-glucanase after 10 days of fermentation. Application of ultrasound waves during fermentation positively affected (p < 0.05) the enzyme activities. The best results for chitinase (51.88 U/g) and ß-1,3-glucanase (39.22 U/g) were obtained with a 50% (w/w) moisture content and 4 h/day ultrasound application for 10 days of fermentation. Increases of 3.6-fold (from 14.37 to 51.88 U/g) and 3.8-fold (from 10.22 to 39.22 U/g) in activities for chitinase and ß-1,3-glucanase, respectively, compared to non-sonicated fermentation, were obtained. Ultrasound technique associated with the SSF process was a promising alternative to increase the production activity of cell wall degrading enzymes: chitinase and ß-1,3-glucanase.

Concentration of exopolysaccharides produced by Fusarium fujikuroi and application of bioproduct as an effective bioherbicide.

Exopolysaccharides are secondary metabolites produced by microorganisms and are a subject of research in many fields of science and industry due to some of their confirmed properties, especially in the pharmaceutical and agrochemical areas. In this context, the objectives of this work were to evaluate the potential of Fusarium fujikuroi for producing exopolysaccharides and to concentrate such compounds in order to increase the herbicidal activity. Exopolysaccharides were produced by submerged fermentation and different concentration methods (membranes, lyophilization, and evaporation) were evaluated. The phytotoxic effects were assessed through absorption assays in detached leaves of Cucumis sativus and evaluated on the seventh day after application. The surface tension was evaluated for each concentration method. The production of exopolysaccharides in the crude broth without concentration was 5.94 g/L. When using the lyophilization method, a maximum yield of exopolysaccharides of 10.64 g/L was obtained. The membranes also presented satisfactory results of exopolysaccharides: 9.60 g/L. The increase of bioherbicidal activity and the lower surface tension are proportionally related to the increase of the concentration of exopolysaccharides.

Enzyme-Catalyzed Production of FAME by Hydroesterification of Soybean Oil Using the Novel Soluble Lipase NS 40116.

The performance of lipase NS 40116, a novel and promising soluble enzyme obtained from modified Thermomyces lanuginosus microorganism, was investigated in the production of biodiesel (fatty acid methyl esters-FAME) by hydroesterification. In order to investigate the potential of the biocatalyst in its soluble form, this work reports the effect of water content and enzyme load, as well as the recovery and reuse of the biocatalyst. A FAME yield of 94.30% after 12 h was achieved at 35 °C by combining 0.50 wt% of lipase, 15 wt% of water, and a methanol:oil molar ratio of 4.5:1. The analysis of the time course reaction suggests that the triacylglycerides (TAGs) are hydrolyzed by the enzyme in a first step, generating free fatty acids (FFAs), followed by the esterification of these FFAs into FAME. In relation to the reusability assays, the lipase kept approximately 90% of its catalytic activity after five cycles of reuse. In this context, the findings of this study demonstrate that lipase NS 40116 can efficiently catalyze hydroesterification reactions under mild conditions, arising as a competitive alternative for biodiesel synthesis.

Concentration of metabolites from Phoma sp. using microfiltration membrane for increasing bioherbicidal activity.

This study is focused on the concentration of fermented broth from Phoma sp. to increase its herbicidal activity. For this purpose, biomolecules produced by submerged fermentation using Phoma sp. were concentrated by hollow fiber microfiltration membranes. The membrane feed was separated into two streams (retentate and permeate) and the crude broth was concentrated to 10, 30, 50, 70 and 90% (relative to the initial volume). The retentate samples were submitted to bioassays (triplicate) for evaluating their phytotoxic effects on five young leaves of species of Cucumis sativus and also on pre-emergence of weeds as Bidens pilosa and Amaranthus retroflexus. The highest herbicidal activity was 80.7% obtained for a concentration of 30% in the retentate fraction. At this condition, the bioherbicide presented severe damage symptoms on the detached leaves of Cucumis sativus if compared to the crude fermented broth. In the pre-emergence of B. pilosa and A. retroflexus, 100% control was obtained for assays performed in a germination chamber. For greenhouse assays using the substrate, the control rate of A. retroflexus was dependent of concentration of bioherbicide. The promising results achieved in the research with membrane separation process allow us to propose and develop further studies for evaluating this technology in the concentration of other metabolites produced by fermentation which also have bioherbicidal activity.

Antifungal and antibacterial activity of extracts produced from Diaporthe schini.

The aim of this work was the production of bioactive metabolites by submerged fermentation from the fungus Diaporthe schini, followed by their extraction, separation and characterization. Different solvents (methanol, dichloromethane and hexane) were used for the extraction of metabolites from the fermentation broth and the extracts obtained were evaluated by in vitro antibacterial and antifungal activity. The separation and characterization of the extract from the hexane extraction was performed by column chromatography and GC-MS, respectively. The extracts had a great inhibitory action on the Gram-positive bacteria Staphylococcus epidermidis and Staphylococcus aureus, on the Gram-negative bacteria Enterobacter aerogenes and Klebsiella pneumoniae and on the fungus Candida krusei. The main metabolites produced were: 13-docosenamide, (Z)-; 2-hexadecene, 3,7,11,15-tetramethyl; 9-octadecenamide and 11-octadecenoic acid. Studies related to the antibacterial and antifungal activities of metabolites extracted from microorganisms are found in the literature. However, works about the identification of metabolites produced by submerged fermentation from Diaporthe schini were not found until the present moment. This work is an initial study where the conditions of the process can be optimized by looking for the production of a specific compound and can be a promising source for obtaining new drugs.

Solid-state fermentation for production of a bioherbicide from Diaporthe sp. and its formulation to enhance the efficacy.

In this study, a bioherbicide was produced by solid-state fermentation (SSF) using Diaporthe sp. Adjuvants were employed in a formulation to enhance the herbicidal activity towards the target (Cucumis sativus). The study was divided into two steps: (1) the fermentation condition for bioherbicide production was assessed; (2) evaluation of different formulations containing palm oil, Tween® 80 and Span® 80, in order to increase phytotoxicity. In step 1, the maximum herbicidal activity (1.23% of the leaves had lesions) was obtained at 25 °C, moisture content of 50 wt%, supplemented with 10 wt% of corn steep liquor and soybean bran and inoculum density of 15 wt%. In step 2, the formulation containing 8.2 wt% of palm oil, 8.2 wt% of Tween® 80 and Span® 80, resulting in an HLB of 12.8 showed the highest phytotoxicity on the leaves. At this condition, dry matter and height of target were reduced about 36% in comparison with control. Diaporthe sp. has the potential to produce molecules with herbicidal activity and the use of adjuvants enhanced three times its efficiency.

Selection, isolation, and identification of fungi for bioherbicide production.

Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture.

Production of bioherbicide by Phoma sp. in a stirred-tank bioreactor.

The objective of this work was to produce an herbicide by submerged fermentation in a stirred-tank bioreactor and to assess the potential herbicidal in pre-emergence, post-emergence, and in a detached leaves of Cucumis sativus var species. wisconsin (cucumber) and Sorghum bicolor (sorghum) species. Fermentations were carried out in a stirred-tank bioreactor with useful volume of 3L. Stirring rate (40, 50, and 60 rpm) and aeration (1, 2 and 3 vvm) were the variables studied for bioherbicide production. Fermented broth was fractioned with different solvents to identify the molecules produced by the fungus in a multi-dimensional gas chromatograph system. Bioherbicide showed 100% inhibition of germination of both species in the pre-emergence tests. From detached leaves tests were verified yellowish lesions in Cucumis sativus and necrotic lesions on leaves of Sorghum bicolor. Post-emergence test presented variation of the phytotoxicity from 25 to 66% for the species C. sativus and from 32 to 58% by S. bicolor. The metabolites produced by submerged fermentation of Phoma sp. presented activity in pre-emergence, post-emergence, and detached leaves of C. sativus and S. bicolor and it could be an alternative in the future for weed control.

Production of cellulolytic enzymes and application of crude enzymatic extract for saccharification of lignocellulosic biomass.

In this study, the optimal conditions for production of cellulolytic enzymes by Trichoderma reesei NRRL-6156 using the solid-state fermentation were assessed in conical flasks and validated in a packed-bed bioreactor. Afterwards, the crude enzymatic extract obtained in the optimized condition was used for hydrolysis of sugarcane bagasse in water and ultrasound baths. The enzyme activities determined in this work were filter paper, exocellulase, endocellulase, and xylanase. The optimized condition for production was moisture content 68.6 wt% and soybean bran concentration 0.9 wt%. The crude enzymatic extract was applied for hydrolysis of sugarcane bagasse, being obtained 224.0 and 229 g kg(-1) at temperature of 43.4 °C and concentration of enzymatic extract of 18.6 % in water and ultrasound baths, respectively. The yields obtained are comparable to commercial enzymes.

Evaluation of fructooligosaccharides separation using a fixed-bed column packed with activated charcoal.

Recent studies have shown that the chromatographic separation of mixtures of saccharides may be improved by making use of activated charcoal, a promising low cost material for the separation of sugars, including fructooligosaccharides. In this work, the development of a methodology to separate fructooligosaccharides from glucose, fructose and sucrose, using a fixed bed column packed with activated charcoal is proposed. The influence of temperature, eluant concentration and step gradients were evaluated to increase the separation efficiency and fructooligosaccharide purity. The final degree of fructooligosaccharide purification and separation efficiency were about 94% and 3.03 respectively, using ethanol gradient concentration ranging from 3.5% to 15% (v/v) at 40°C. The fixed bed column packed with the activated charcoal was shown to be a promising alternative for sugar separation, mainly those rich in fructooligosaccharides, leading to solutions of acceptable degrees of purification.

Effects of Solvent Diols on the Synthesis of ZnFe2O4 Particles and Their Use as Heterogeneous Photo-Fenton Catalysts.

A solvothermal method was used to prepare zinc ferrite spinel oxide (ZnFe2O4) using ethylene glycol and 1,4 butanediol as solvent diols, and the influence of diols on the physical properties of ZnFe2O4 particles was investigated. The produced particles were characterized by X-ray powder diffraction (XRD), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and nitrogen adsorption isotherms, and the catalytic activity for the organic pollutant decomposition by heterogeneous photo-Fenton reaction was investigated. Both solvents produced particles with cubic spinel structure. Microporous and mesoporous structures were obtained when ethylene glycol and 1,4 butanediol were used as diols, respectively. A higher pore volume and surface area, as well as a higher catalytic activity for the pollutant degradation were found when 1,4 butanediol was used as solvent.