Andrea de Souza Monteiro

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 75u2009g/L biodiesel residue and 5u2009g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58u2009g/L biodiesel refinery residue and 4.6u2009g/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%). 1u2009H 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.

The use of acetonitrile as the sole nitrogen and carbon source by Geotrichum sp. JR1

A yeast strain identified as Geotrichum sp. JR1 was able to use acetonitrile as the sole carbon and nitrogen source. The strain grew in 0.5 to 2M acetonitrile. Ammonia generation as enzymatic product during the strain growth indicates the presence of an acetonitrile degrading enzyme. Acetic acid and acetamide were detected during assays with the resting cells cultivated in acetonitrile, indicating the presence of nitrile and amide degrading enzymes. This paper is the first to describe the use of acetonitrile as the sole carbon and nitrogen source by a yeast.

Isolation and identification of Candida species in patients with orogastric cancer: susceptibility to antifungal drugs, attributes of virulence in vitro and immune response phenotype

BackgroundBecause of the inherent immunosuppression of cancer patients opportunistic infections by Candida spp, occur frequently. This study aimed to identify Candida species in the oral mucosa of 59 patients with orogastric cancer (OGC) and to analyze the immunological phenotype of these patients.MethodsThe yeasts were identified by MALDI-TOF mass spectrometry (MS). For all isolates, we performed phospholipases and proteinases assays, in vitro adherence to buccal epithelial cells (BEC), minimum inhibitory concentration of antifungal drugs and determined the cytokine profile by Cytometric Bead Array flow citometry assay.ResultsC. albicans was the most prevalent species in OGC patients (51.6xa0%) and control group (66.7xa0%). Candida spp. strains isolated from OGC patients exhibited better adherence to BEC (pu2009=u20090.05) than did the control group. Phospholipases production by Candida strains from OGC patients was lower (51.6xa0%) than in the control group (61.9xa0%). Proteinases were detected in 41.9xa0% and 4.8xa0% of the yeasts from OGC patients and control group, respectively. Significant differences were found in the serum of OGC patients compared to the control group for IL-2, IL-10, TNF-α, IFN-γ and IL-17.ConclusionsThe results of this work suggest increased virulence of yeasts isolated from OGC patients and, that this may interfere with the immune phenotype.

Phenotypic and molecular detection of the bla KPC gene in clinical isolates from inpatients at hospitals in São Luis, MA, Brazil

BackgroundBacteria that produce Klebsiella pneumoniae carbapenemases (KPCs) are resistant to broad-spectrum β-lactam antibiotics. The objective of this study was to phenotypically and genotypically characterize the antibiotic susceptibility to carbapenems of 297 isolates recovered from clinical samples obtained from inpatients at 16 hospitals in São Luis (Maranhão, Brazil).MethodsThe study was conducted using phenotypic tests and molecular methods, including polymerase chain reaction (PCR), sequencing and enterobacterial repetitive intergenic consensus (ERIC)-PCR. The nonparametric chi-square test of independence was used to evaluate the associations between the bacterial blaKPC gene and the modified Hodge test, and the chi-square adherence test was used to assess the frequency of carbapenemases and their association with the blaKPC gene.ResultsThe most frequently isolated species were Acinetobacter baumannii (nu2009=u2009128; 43.0%), K. pneumoniae (nu2009=u200975; 25.2%), and Pseudomonas aeruginosa (nu2009=u200942; 14.1%). Susceptibility assays showed that polymixin B was active against 89.3% of the bacterial isolates. The Acinetobacter spp. and K. pneumoniae strains were susceptible to amikacin and tigecycline, and Pseudomonas spp. were sensitive to gentamicin and amikacin. Among the 297 isolates, 100 (33.7%) were positive for the blaKPC gene, including non-fermentative bacteria (A. baumannii) and Enterobacteriaceae species. Among the isolates positive for the blaKPC gene, K. pneumoniae isolates had the highest positivity rate of 60.0%. The blaKPC gene variants detected included KPC-2, which was found in all isolates belonging to species of the Enterobacteriaceae family. KPC-2 and KPC-3 were observed in A. baumannii isolates. Importantly, the blaKPC gene was also detected in three Raoultella isolates and one isolate of the Pantoea genus. ERIC-PCR patterns showed a high level of genetic diversity among the bacterial isolates; it was capable of distinguishing 34 clones among 100 strains that were positive for blaKPC and were circulating in 11 of the surveyed hospitals.ConclusionsThe high frequency of the blaKPC gene and the high degree of clonal diversity among microorganisms isolated from patients from different hospitals in São Luis suggest the need to improve the quality of health care to reduce the incidence of infections and the emergence of carbapenem resistance in these bacteria as well as other Gram-negative pathogens.

Phytochemical Characterization of Terminalia catappa Linn. Extracts and Their antifungal Activities against Candida spp.

Terminalia catappa Linn bark is used to treat dysentery by various populations in Southeast Asian countries, and its leaves have also been used in traditional medicine to treat hepatitis in India and the Philippines. Here, the antifungal actions of crude hydro-alcoholic extract (TcHE) and fractions from T. catappa leaves were assessed via the agar diffusion and microdilution tests on Candida reference strains and clinical isolates from patients with acquired immunodeficiency syndrome (AIDS). Additionally, the potential cytotoxic effects of TcHE were assessed on cultured human peripheral blood mononuclear cells (PBMC). T. catappa fractions and sub-fractions were analyzed by gas chromatography coupled to mass spectrometry with electron impact (GC/MS/EI), high-performance liquid chromatography coupled to mass spectrometry “electrospray” ionization in positive mode (HPLC/MS/MS/ESI+) and hydrogen nuclear magnetic resonance (1HNMR). TcHE and its fractions were able to inhibit the growth of all tested Candida strains with the n-butanol (FBuOH) fraction presenting the best antifungal activity. Testing of different FBuOH sub-fractions (SF) showed that SF10 was the most active against Candida spp. Fractioning of SF10 demonstrated that 5 out of its 15 sub-fractions were active against Candida spp., with SF10.5 presenting the highest activity. Chemical analysis of SF10 detected hydrolysable tannins (punicalin, punicalagin), gallic acid and flavonoid C-glycosides. Overall, the results showed that T. catappa L. leaf extract, fractions and sub-fractions were antifungal against Candida spp. and may be useful to treat diseases caused by this fungus.