Vera Lúcia dos Santos

Biodegradation of phenol by a filamentous fungi isolated from industrial effluents: identification and degradation potential

Thirty filamentous fungal strains were isolated from effluents of a stainless steel industry (Minas Gerais, Brazil) and tested for phenol tolerance. Fifteen strains of the genera Fusarium sp., Aspergillus sp., Penicillium sp. and Graphium sp. tolerants up to 10 mM of phenol were selected and tested for their ability to degrade phenol. Phenol degradation was a function of strain, time of incubation and initial phenol concentration. FIB4, LEA5 and AE2 strains of Graphium sp. and FE11 of Fusarium sp. presented the highest percentage phenol degradation, with 75% degradation of 10 mM phenol in 168 h for FIB4. A higher starting cell density of Graphium sp. FIB4 lead to a decrease in the time needed for full phenol degradation and increased the phenol degradation rate. All strains exhibited activity of catechol 1,2-dioxygenase and phenol hydroxylase in free cell extracts obtained from cells grown on phenol, suggesting that catechol was oxidized by the ortho type of ring fission. These data reported demonstrate the prospect after the application of filamentous fungal strains in protecting the environment from phenol pollution.

Phenol degradation by Aureobasidium pullulans FE13 isolated from industrial effluents

The degradation of phenol (2-30 mM) by free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13 isolated from stainless steel effluents was studied in batch cultures with saline solution not supplemented with nutrients or yeast extract. The rate at which the immobilized cells degrade phenol was similar to the rate at which the suspended cells could degrade phenol, for a concentration of up to 16 mM of phenol. The maximum phenol volumetric degradation rate for 16 mM phenol was found to be 18.35 mg l(-1)h(-1) in the assays with free cells and 20.45 mg l(-1)h(-1) in the assays with alginate-immobilized cells, 18 mM phenol and cellular concentration of 0.176 g/l. At concentrations higher than this, an inhibitory effect was observed, resulting in the lowering of the phenol degradation rates. The immobilization was detrimental to the catechol 1,2-dioxygenase activity. However, the immobilized cells remained viable for a longer period, increasing the efficiency of phenol degradation. The yeast showed catechol 1,2-dioxygenase activity only after growth in the phenol, which was induced at phenol concentrations as low as 0.05 mM and up to 25 mM at 45 h of incubation at 30 degrees C. Phenol concentrations higher than 6mM were inhibitory to the enzyme. Addition of glucose, lactate, succinate, and benzoate reduced the rate at which phenol is consumed by cells. Our results suggest that inoculants based on immobilized cells of A. pullulans FE13 has potential application in the biodegradation of phenol and possibly in the degradation of other related aromatic compounds.

Identification and characterization of bioemulsifier-producing yeasts isolated from effluents of a dairy industry.

New bioemulsifier-producing yeasts were isolated from the biological wastewater treatment plant of a dairy industry. Of the 31 bioemulsifier-producing strains, 12 showed emulsifying activity after 2months of incubation, with E(24) values ranging from 7% to 78%. However, only Trichosporon loubieri CLV20, Geotrichum sp. CLOA40, and T. montevideense CLOA70 exhibited high emulsion-stabilizing capacity, with E(24) values of 78%, 67%, and 66%, respectively. These isolates were shown to induce a strong emulsion stabilizing activity rather than the reduction of the interfacial tension. These strains exhibited similar growth rates in the exponential growth phase, with a clear acceleration after 24h and stabilization of the activity after 144h. Emulsification and stability properties of the bioemulsifiers were compared to those of commercial surfactants after the addition of NaCl and exposure to temperature of 100 degrees C. The compounds produced by the isolates appeared to be lipid-polysaccharide complexes. Gas chromatograph analysis of the lipidic fraction of the bioemulsifiers from CLV20, CLOA40, and CLOA70 shows the prevalence of (9Z,12Z)-octadeca-9,12-dienoic acid, in concentrations of 42.8%, 25.9%, and 49.8%, respectively. The carbohydrate composition, as determined by GC-MS of their alditol acetate derivatives, showed a predominance of mannose, galactose, xylose and arabinose.

Inhibition of Candida albicans CC biofilms formation in polystyrene plate surfaces by biosurfactant produced by Trichosporon montevideense CLOA72.

This study evaluated the effects of glycolipid-type biosurfactant produced by Trichosporon montevideense CLOA72 in the formation of biofilms in polystyrene plate surfaces by Candida albicans CC isolated from the apical tooth canal. Biofilm formation was reduced up to 87.4% with use of biosurfactant at 16 mg/ml concentration. It has been suggested that the interaction with the cell or polystyrene plate surface could ultimately be responsible for these actions. Therefore, the interaction of C. albicans CC cells with the biosurfactant, as well as the corresponding thermodynamic parameters, have been determined by isothermal titration calorimetry and zeta potential measurements. This process is endothermic (((int)H°=+1284±5 cal/mg OD(600)) occurring with a high increase of entropy (T((int)S°=+10635 cal/mg OD(600)). The caloric energy rate data released during the titulation indicates saturation of the cell-biosurfactant at 1.28 mg/ml OD(600). Also, the zeta potential of the cell surface was monitored as a function of the biosurfactant concentration added to cell suspension showing partial neutralization of net surface charge, since the value of zeta potential ranged from -16 mV to -6 mV during the titration. The changes of cell surface characteristics can contribute to the inhibition of initial adherence of cells of C. albicans in surface. The CMC of the purified biosurfactant produced from T. montevideense CLOA72 is 2.2 mg/ml, as determined both by ITC dilution experiments and by surface tension measurements. This biomolecule did not presented any cytotoxic effect in HEK 293A cell line at concentrations of 0.25-1 mg/ml. This study suggests a possible application of the referred biosurfactant in inhibiting the formation of biofilms on plastic surfaces by C. albicans.

Demulsifying properties of extracellular products and cells of Pseudomonas aeruginosa MSJ isolated from petroleum-contaminated soil

A strain of Pseudomonas aeruginosa isolated from a site contaminated with refined oil products exhibited demulsification capabilities against Tween 80-Span 80 stabilized oil-in-water (O/W), Tween 80-stabilized water-in-oil (W/O) model emulsions (kerosene-water), and an industrial emulsion (Daido Dairoll PA-5A). GC-MS analysis confirmed the presence of fatty acids and carbohydrates in the extracellular biodemulsifier. The demulsifying activity of cells and culture supernatants was favored by growth in media containing 1% diesel oil. There was a correlation between culture age, de-emulsification and cellular hydrophobicity, and highest activities were observed for cells and supernatants from 96-h cultures. Activity increased with addition of up to 60 mg cells or 300 μL supernatant to emulsions. The activity was relatively stable at 20-40 °C and to freezing, but was reduced by 69% by washing the cells with chloroform-methanol-water. This demulsifier has potential for application in biotreatment of emulsified oily wastewaters to promote recovery and/or degradation of oil.

Characterization of new biosurfactant produced by Trichosporon montevideense CLOA 72 isolated from dairy industry effluents.

The yeast strain CLOA 72 isolated from the effluent of a dairy industry in Brazil and identified as Trichosporon montevideense, was able to grow and produce a glycolipid biosurfactant when cultured on a mineral medium (MM) with sunflower oil as the carbon source. Biosurfactant production was partially growth‐associated and maximal emulsification activity was observed at 144 h of cultivation (78.92%). The biosurfactant purified by precipitation with ethanol showed 78.66% emulsifying activity when used in concentrations above 4.5 mg/ml and was able to reduce the surface tension of water to values below 44.9 mN/m. The critical micellar concentration (CMC) was found to be 2.2 mg/ml. The highest emulsifying activity (E24) has been observed with vegetable oils, toluene, kerosene, isooctane, cyclohexane, hexane, diesel oil and hexadecane as compared to mineral oil and oleic acid. The biosurfactant also showed good stability during exposure to 100 °C for different periods of time (10 to 60 min), to high salinity (30% of NaCl, KCl and NaHCO3), and to a wide range of pH values (1–10). The biosurfactant purified by gel filtration chromatography is a glycolipid, with lipid portion containing 16.03% (9Z)‐octadec‐9‐enoic acid, 14.92% hexadecanoic acid, and 9.63% (E) octadec‐9‐enoic acid and the carbohydrate portion containing mannose (35.29%), xylose (41.99%), arabinose (17.47%), and glucose (5.25%). (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2

BackgroundThe microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes.ResultsTrichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide’s and long chain aliphatic groups in the bioemulsifier molecule.ConclusionsThe biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.

Degradation of phenol by Trichosporon sp. LE3 cells immobilized in alginate.

The degradation of phenol by freely suspended cells of Trichosporon sp. LE3 and alginate‐immobilized cells was studied in batch culture. The alginate concentration (2 or 4%) and the cross‐linking salt used (BaCl2 or CaCl2) affected the rate and percentage of phenol degradation. The highest values were obtained for immobilized cells at 2% calcium alginate, although complete degradation of 15 and 18 mM phenol was not observed. When the cell concentrations in the assays were doubled, the 2% calcium alginate‐immobilized cells were able to degrade up to 30 mM phenol in less than 120 hours, although the free cells did not completely degrade phenol at concentrations above 20 mM. The maximum phenol degradation rate was a strong function of initial phenol concentrations, being the highest values being observed for 20 mM phenol.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

Ethanol production from macaúba (Acrocomia aculeata) presscake hemicellulosic hydrolysate by Candida boidinii UFMG14

Yeasts capable of growth on xylose were isolated from macaúba (Acrocomia aculeata) fruit, a Brazilian palm tree with great potential for use as biodiesel feedstock production. Candida boidinii UFMG14 strain achieved the highest ethanol production (5 g/L) and was chosen to ferment macaúba presscake hemicellulosic hydrolysate (MPHH). The MPHH was produced by the first time in this work and the resultant fivefold concentrate showed considerable sugar content (52.3 and 34.2 g/L xylose and glucose, respectively) and low furfural (0.01 g/L) and hydroxymethylfurfural (0.15 g/L) concentrations. C. boidinii UFMG14 fermentation was evaluated in supplemented and non-supplemented MPHH containing either 10 or 25 g/L of xylose. The maximum ethanol production (12 g/L) was observed after 48 h of fermentation. The ethanol yield was significantly affected by supplementation and concentration of MPHH while ethanol productivity was affected only by MPHH concentration. This is the first study demonstrating theC. boidinii potential for ethanol production from hemicellulose byproducts.