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BACKGROUND The main objective of this study was to examine the growth and phenol degradation kinetic parameters for bacteria isolated from activated sludge that could register growth on phenol as high as 1410 mg L-1. RESULTS Phenol-degrading bacteria of the Alcaligenes, Providencia, Acinetobacter and Aeromonas genera were prevalent in the activated-sludge tank. The degradation rate was affected by the phenol concentration; the B9-1 and B8-1 Alcaligenes faecalis strains showed rates of 0.037 and 0.038 g L-1 h-1 when exposed to up to 940 mg L-1 phenol, respectively. The B6-2, G1, and H1-1 A. faecalis strains showed rates of 0.036, 0.042 and 0.036 g L-1 h-1, respectively, when exposed to up to 1128 mg L-1, and Acinetobacter johnsonii D1 and A. faecalis D3 strains showed rates of 0.058 and 0.049 g L-1 h-1, respectively, when exposed to 1410 mg L-1 phenol. The growth kinetic parameters adjusted by genetic algorithm (GA) and the Haldane equation ranged within μm = 0.12–0.55 h-1, μmax = 0.07–0.29 h-1, Ks = 25–483.8 mg L-1, and Ki = 153.7–2582.6 mg L-1. CONCLUSIONS The high values of μmax, Ki, and YX/S adjusted by GA showed that the studied strains are able to tolerate and degrade higher phenol concentrations compared with levels reported for single bacterial strains. © 2014 Society of Chemical Industry

Kraft pulp mill foul condensates were treated in a 4-L membrane bioreactor, which was fed continuously with a chemical oxygen demand (COD) load of 5.12 kg COD/m3/day, and a hydraulic retention time (HRT) of 19 h. During a period of 60 days, three temperatures were tested 35, 45 and 55 °C. Very high COD, total reduced sulphur (TRS) and methanol removals were obtained in the mesophilic temperature range. As the temperature increased, a decrease in efficiency was observed, although it could still considered to be high. A decrease in respiratory activity from 19.98 mg O2/g TSS/h at 35 °C to 12.87 mg O2/g total suspended solids (TSS)/h obtained at 55 °C, followed by a decrease in the TSS concentration in the bioreactor, suggested that the biomass is thermotolerant. The results of this research showed that the treatment of foul condensates from kraft pulp mills at high temperatures using a membrane bioreactor was shown to be technically feasible and it has good potential for industrial application.

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. Uma linhagem de levedura identificada como Geotrichum sp. JR1 foi capaz de utilizar acetonitrila, em concentrações de 0,5 a 2M, como única fonte de carbono e de nitrogênio. A geração de amônia durante o crescimento do microrganismo indica a presença de sistema enzimático capaz de degradar acetonitrila. Durante os ensaios enzimáticos, com células cultivadas em acetonitrila, foram detectados ácido acético e acetamida como produtos indicando a presença de sistema enzimático capaz de degradar acetonitrila e acetamida. Este trabalho é o primeiro a descrever a utilização de acetonitrila como única fonte de carbono e de nitrogênio por uma levedura.

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.

In this work, we show that the fungal strain Graphium sp. FIB4 was able to use phenol as the sole carbon source. Higher degradation of phenol was accomplished by alginate-immobilized mycelial mass than by mycelial suspensions of Graphium sp. FIB4. Free mycelium exhibited higher degradation rates when compared with the alginate-immobilized mycelium in the presence of 14 mM of phenol or less. Above this concentration, degradation rates by free mycelium decreased and the immobilized mycelium showed higher values. The maximum degradation rate for 8 mM phenol was found to be 20.13 mg/l x h by free mycelia and 16.24 mg/l x h by immobilized mycelial mass in the presence of 18 mM phenol. When the fungus was grown on medium without phenol, catechol 1,2-dioxygenase activity was not detected. This enzyme activity was induced at phenol concentrations as low as 0.05 mM and up to 6 mM at 24 h incubation at 30 degrees C, suggesting that catechol was oxidized by the ortho type of ring fission. Addition of glucose reduced phenol consumption rate, and both substrates were used simultaneously. Glucose concentrations higher than 0.075% repressed the induction of phenol oxidation by Graphium sp. FIB4 grown on glucose. But glucose did not fully repress utilization of phenol by phenol-pre-induced cells. Immobilization and addition of calcium and barium ions were detrimental to the stability of catechol 1,2-dioxygenase activity and phenol degradation by Graphium sp. FIB4.

Nitrile degradation by Candida guilliermondii CCT 7207 using free and immobilized cell systems was compared. Different specific growth rates were observed for immobilized (mumax=0.021 h(-1)) and the free cells (mumax=0.029 h(-1)). The maximum specific rate of acetic acid formation was 0.387 h(-1) and 0.266 h(-1) for free and immobilized cells, respectively. Cell adhesion to the support materials was confirmed by scanning electron microscopy. When immobilized, the yeast was able to use high nitrile and amide concentrations (aliphatic and aromatic) as nitrogen sources. The results suggest that C. guilliermondii CCT 7207 presents a physiological pattern potentially useful for the bioremediation of polluted environments or for the bioproduction of amides and organic acid of high commercial value.

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.

Yeast strains of the genera Aureobasidium, Rhodotorula and Trichosporon were isolated from stainless steel effluents and tested for their ability to utilize phenol as the sole carbon source. Fourteen strains grew in the presence of up to 10 mm phenol. Only the strain Trichosporon sp. LE3 was able to grow in the presence of up to 20 mm phenol. An inhibitory effect was observed at concentrations higher than 11 mm, resulting in reduction of specific growth rates. Phenol degradation was a function of strain, time of incubation and initial phenol concentration. All strains exhibited activity of catechol 1,2-dioxygenase and phenol hydroxylase in free cell extracts from cells grown on phenol, suggesting that catechol was oxidized by the ortho type of ring fission. Addition of glucose and benzoate reduced the phenol consumption rate, and both substrates were used simultaneously. Glucose concentrations higher than 0.25% inhibited the induction of phenol oxidation by non-proliferating cells and inhibited phenol oxidation by pre-induced cells.

A full factorial design leading to 20 sets of sorption runs was conducted to study the influence of four variables (bleaching earth and biomass concen- trations, pH, and sorption time) on the iron, nickel, and chromium removal from stainless steel effluent using waste biomass from a beverage industry. Similar factor effects and interactions were found for each metal involved in this biosorption study, and the main factors were pH (positive effect) and biomass concentration (negative effect). Response surface methodology was adopted and an empirical linear polynomial model constructed on the basis of the specific uptake (mg of metal/g of biomass as dry weight) for each metal species. Under optimized process conditions (pH 4.0, biomass concentration of 2.0 g/L, absence of Celite), uptake values of 155 mg of Fe/g, 38 mg of Cr/g, and 0.4 mg of Ni/g were achieved after 3 h. This corresponded to a reduction in heavy metals concentration of approx 94% for Cr, 57% for Fe, and 25% for Ni.

The yeast Cryptococcus sp. UFMG-Y28 can utilize benzonitrile as a nitrogen and possible additional carbon source. The kinetics of growth on Yeast Carbon Base (YCB) added of benzonitrile as sole nitrogen source showed that benzonitrile was metabolized to benzoic acid and ammonia. Liquid chromatography analysis indicated that Cryptococcus sp. UFMG-Y28 metabolized 12 mM benzonitrile to 10 mM benzoic acid. Resting cells cultivated on YCB-propionitrile medium showed nitrilase activity against benzonitrile. This strain appears to be promising for bioconversion of nitriles to high value acids and for bioremediation of sites contaminated with aliphatic and aromatic nitriles.

Different encapsulation matrices were tested for immobilized cells of Candida guilliermondii UFMG-Y65 used for acetonitrile degradation. Acetonitrile degradation by free cells and cells immobilized in Ba-alginate, kappa-carrageenan and citric pectin was studied. The rate of acetonitrile degradation was monitored for 120 h by measuring yeast growth and ammonia concentration. Different alginate concentrations did not affect cell viability, but the period of incubation in BaCl2 solution reduced the number of viable cells. Likewise, the gel nature and the matrix structure of the support resulting from the cell immobilization conditions were of fundamental importance for biocatalyst activity and performance, affecting substantially the patterns of microbial growth and enzymatic activity. Alginate-immobilized cells degraded acetonitrile more efficiently than kappa-carrageenan or citric pectin-immobilized cells. Na degradação de acetonitrila, foram testadas células livres de Candida guilliermondii UFMG-Y65 e imobilizadas em diferentes suportes, quais sejam, Ba-alginato, kapa-carrageno e pectina cítrica. A velocidade de degradação da acetonitrila foi monitorada por 120 h, mediante o crescimento da levedura e geração de amônia. Diferentes concentrações de alginato não afetam a viabilidade das células; mas o período de incubação, em solução de BaCl2, reduziu o número de células vivas. Da mesma forma, a natureza do gel e a estrutura da matriz do suporte, ambas resultantes das condições de imobilização das células, foram de fundamental importância para a atividade catalisadora e sua performance; afetando assim, os padrões de crescimento microbiano e a de atividade enzimática. As células imobilizadas em alginato degradaram acetonitrila com maior eficácia do que as imobilizadas em kapa-carrageno ou as células imobilizadas em pectina cítrica.

Yeast strains from the genera Candida, Debaryomyces, Aureobasidium, Geotrichum, Pichia, Rhodotorula, Tremella, Hanseniaspora, and Cryptococcus were isolated from samples of a gold mine from liquid extraction circuit. These strains were tested for their ability to utilize acetonitrile at 12 mM as the sole nitrogen source. The yeasts that grew using acetonitrile at 12 mM were tested in the presence of acetonitrile, isobutyronitrile, methacrylnitrile, and propionitrile at concentrations of 12, 24, 48, 97, and 120 mM. One strain was selected for each nitrile and the concentration of nitrile in which the best growth occurred. Cryptococcus sp. strain UFMG-Y28 had a better growth on 120 mM propionitrile and 97 mM acetonitrile, Rhodotorula glutinis strain UFMG-Y5 on 48 mM methacrylnitrile, and Cryptococcus flavus strain UFMG-Y61 on 120 mM isobutyronitrile. The utilization of different nitriles and amides by yeast strains involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.

Aspergillus niger was grown in an industrial gold mining solution containing cyano-metal complexes. Gold, silver, copper, iron and zinc were accumulated, and the major mechanism of metal uptake involved metal precipitation on the cell surface. This could be achieved by the extensive application of instrumental analyses combined to the study of the chemical composition of the solutions, before and after contact with the fungus.

Summary Eighty seven yeast strains representing 34 species isolated from Parahancornia amapa fruit and associated Drosophila flies collected in the Brazilian Amazon rain forest, were screened for proteinase production. Proteolytic activity was tested through casein hydrolysis in solid medium supplemented with 0.5% casein and glucose. Among 23 strains, 18 from genus Candida and 5 from Pichia were caseinolytic and produced proteinases in yeast carbon base liquid medium supplemented with casein 0.01%. The proteolytic activity was tested on pH ranging from 2.0 to 9.0 in correspondance to the pH of the cultures media in which the yeasts were grown. Six highly proteolytic strains: Candida parapsilosis AP153A, C. krusei AP176, C. sorbosa DR215, C. sorbosa AP259, C. valida AP209A and C. sorboxylosa AP287 were selected and the pH optima of production and the proteolytic activity were determined. In general the secretion of proteinase was maximum throughout the exponential and the stationary phases. Greater production occurred in acidic culture and high activity was observed at pH 3.0, 4.0 and 5.0.

Strains of Saccharomyces spp. from tropical substrates tolerated temperatures up to 40 ° C, sucrose concentrations up to 50% (w/v) and ethanol concentrations up to 20 g/L in fermentation conditions. Strain TD200 tolerated 20 g/l of ethanol. The ethanol produced by strain DR1459 was comparable to that of industrial strain HTYM-81. These strains have potential use for the production of fuel alcohol.

Chemical formation of a glucose–cyanide complex was necessary for metabolic degradation of cyanide at concentrations up to 50.0 mg/L by a strain of Escherichia coli isolated from gold extraction circuit liquids. Ammonia accumulating during the culture log phase as the sole nitrogen by-product was further utilized for bacterial growth. Washed (intact) cells, harvested at different periods of bacterial growth on cyanide, consumed oxygen in presence of cyanide. These findings suggest that metabolism of cyanide involved a dioxygenase enzyme that converted cyanide directly to ammonia, without the formation of cyanate.Key words: cyanide, cyanide metabolism, cyanide oxygenase, Escherichia coli.

Some physical and chemical properties ofα-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) produced in semisolid fermentation byCandida fennica FTPT-1829,C. famata FTPT-1539 andC. fennica FTPT-8903 were determined. The optimum temperature values were 42, 44, 48 °C and 60, 50, 50 °C forα-amylase and glucoamylase excreted byC. fennica 8903,C. fennica 1829 andC. famata 1539, respectively. The optimum pH values for all strains were 5.0 and 6.0 forα-amylase and glucoamylase, respectively. The degradation of pullulan by all the yeast species indicates debranching activity.

TheRhodotorula rubra strain isolated from waste waters of a gold mining plant has demonstrated the ability to grow in the presence of cyanide. The maintenance of this strain in complex organic media leads to a loss of this ability. To preserve the cyanide resistance ofR. rubra we tested the following maintenance methods: subculturing in sterile distilled water, freezing at −20, −40, −70°C, liquid nitrogen freezing and the paper replica method. The ability to grow in the presence of cyanide was preserved and a higher viability level was observed for cells maintained frozen at −70°C, in liquid nitrogen and by the paper replica method. Preservation in distilled water resulted in the lowest viability after twelve months of storage.

An evaluation of the efficiency of treatment of kraft mill foul condensates in a membrane bioreactor was carried out in the laboratory. Efficiency and rate of methanol removal were quantified at operating temperatures of 35, 45 and 55 degrees C. The structure of the bacterial community present in the reactor biomass at the different operating temperatures was evaluated by in situ hybridization of the biomass samples with fluorescently-labelled probes (FISH) targeting the Eubacteria, the alpha, beta and gamma subclasses of the Proteobacteria, the low G + C content Gram-positive bacteria (Bacillus spp.), while community function was evaluated by in situ hybridization with a methanol dehydrogenase gene (mxaF) probe. Methanol removal efficiency decreased from 99.4 to 92%, and removal rate from 2.69 mg MeOH/l x min to 2.49 mg MeOH/l x min when the operating temperature was increased from 35 to 55 degrees C. This decrease in methanol removal was accompanied by a decrease (from 58% to 42%) in the relative proportion of cells that hybridized with the mxaF probe. The relative proportion of Bacillus spp. increased from 5 to 20% while the proportion of members of the alpha subclass of Proteobacteria decreased from 16% to 6% when the bioreactor operating temperature was raised from 35 to 55 degrees C. The relative proportions of bacteria belonging to the beta (22-25%) and gamma (18-20%) subclasses of the Proteobacteria remained relatively constant regardless of operating temperature. Proteobacteria (alpha, beta and gamma subclasses) and Bacillus spp. represented 61, 67 and 71% of the Eubacteria in the biomass sampled at 35, 45 and 55 degrees C, respectively. The FISH technique was shown to be an efficient method for detection of both structural and functional changes in the bacterial communities that could be related to efficiency of methanol removal in a membrane bioreactor operating at different temperatures.

Yeast cells immobilized by entrapment in Ba-alginate gel were investigated for growth pattern and respiratory activity. The oxygen uptake rates (OUR) of cells entrapped in gels with 4% alginate were 5.2 and 23% lower than the OUR of 2% alginate and free cells, respectively. The mass-transfer resistance offered by the matrix and growth of the entrapped cells determine a gradient of nutrients throughout the gel which is responsible for both a lower specific growth rate of immobilized cells with respect to that of free ones, and a heterogeneous biomass distribution, with progressively increasing cellular density from the inside to the outside of the matrix. Gel-matrix polymer concentration affected the maximum oxygen uptake of immobilized growing yeast cells.

Candida guilliermondii UFMG-Y65, isolated from a gold mine, was able to utilize different nitriles and the corresponding amides as sole source of nitrogen, at concentrations up to 2 M. Resting cells cultivated on YCB-acetonitrile medium showed nitrile hydrolyzing enzyme activities against acrylonitrile and benzonitrile. These enzymes were inducible and intracellular; the optimum pH was 7.0-8.0, and the optimum temperature 25°C-30°C. Liquid chromatographic analysis indicated that C. guilliermondii UFMG-Y65 metabolized 12 mM benzonitrile to 11 mM benzoic acid and 10 mM acrylonitrile to 7.9 mM acrylic acid. The results suggest that C. guilliermondii UFMG-Y65 may be useful for the bioproduction of amides and acids, and for the bioremediation of environments contaminated with nitriles.Key words: Candida guilliermondii, nitrile hydrolyzing enzyme, amidase, nitriles, amides.

Yeasts (228) isolated for natural habitats were screened for their ability to produce amylases in semisolid medium of wheat bran. Strains of Aureobasidium pullulans, Candida famata, and Candida kefyr showed high enzymatic activity for α-amylase, glucoamylase, and debranching enzyme. Key words: Aureobasidium, Candida, amylolytic yeasts, α-amylase, glucoamylase.

A variant of a yeast strain identified as Candida famata isolated from gold mine effluent was able to grow on acetonitrile, acrylonitrile, butyronitrile, isobutyronitrile, methacrylnitrile, propionitrile, succinonitrile, valeronitrile, acetamide, isobutyamide, and succinamide as sole nitrogen source, after acclimatization. The yeast grew on acetonitrile and acetamide at concentrations up to 4%. The utilisation of acetonitrile and acetamide by the C. famata strain probably involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.

The production of α-amylase was increased by protoplast fusion of auxotrophic mutants of Candida fennica FTPT-8903. One prototrophic fusant was 90% and 32% more efficient in producing α-amylase in semi-solid and liquid fermentation, respectively, than the parental strains. Protoplast fusion did not significantly stimulate the synthesis of glucoamylase in the fusants.

The capacity of waste biomasses from sugar-cane aguardente, a traditional Brazilian spirit, for metal biosorption was assessed. Free biomass and biomass immobilized onto chitin and Dowex (ion-exchange resin) were utilized to remove chromium, iron and nickel from both synthetic solutions and stainless steel effluents. The best performance in terms of metal sorbed was observed in with free biomass, with the following adsorption capacity: 70% chromium, 50% iron and 20% nickel at pH 4.0.

The viability of the yeast Rhodotorula rubra, isolated from liquid samples of gold-mine effluents, was not affected by the presence of 11.52 mM cyanide. The yeast was able to utilize ammonia, generated from abiotic cyanide degradation in the presence of reducing sugars, in aerobic culture at pH 9.0. These physiological characteristics encourage studies with mixed cultures of cyanide-degrading organisms, using this yeast as an assimilator of ammonia.

An Aspergillus niger strain accumulated gold and silver from solution containing high cyanide concentrations. Our results show that A. niger is of potential use for the removal of metals and/or the recovery of valuable metals from cyanide-containing solutions.