The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF-and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20-22%) and gamma (11-12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.

Penicillium griseoroseum was grown in bioreactors on mineral medium supplemented with yeast extract and sucrose. The influence of inoculum and carbon source concentrations, aeration and pH on pectin lyase (PL) production, as well as the capacity of P. griseoroseum to produce PL when grown on sugar cane syrup as carbon source were evaluated. Inoculum concentration did not influence PL production. Production was higher in non-aerated than in aerated medium. The best results were obtained using 60 mM sucrose at pH 6.3-7.2. Production using cane syrup 25% (v/v), without yeast extract supplement, was equal to that obtained under the conditions cited above.

Microbial colonies were replicated on YNB® agar plates overlaid with soft agar containing the glucose-oxidase/peroxidase (BIOTROLâ) system. The pink color developed around the colonies was the result of the reaction of the glucose generated by the extracellular hydrolysis of lactose by b-galactosidase, indicating secretion of this enzyme. This method proved to be very convenient for testing hundreds of colonies grown on agar plates for b-galactosidase secretion by microbial cells.

Pectin lyase (PL) from Penicillium griseoroseum can be induced by xanthine, theobromine, theophylline and especially by caffeine and hypoxanthine (5 mmol l(-1) With 0.01% Beast extract (YE)). For caffeine and hypoxanthine, PL activity was, respectively, 5.2 and 3.7 times higher than with YE alone. The simultaneous addition of caffeine or hypoxanthine (5 mmol l(-1)) and YE (0.1%) had a synergistic effect on PL activity as compared to the addition of these substances alone (0.2% YE; 10 mmol l(-1) caffeine; 10 mmol l(-1) hypoxanthine). Increasing caffeine concentrations (0-10 mmol l(-1)) for a constant YE, content of 0.01%, resulted in an increase in PL activity and a decrease in mycelial mass. For a constant caffeine concentration (5 mmol l(-1)) and increasing YE contents (0-0.2%), a higher PL activity and mycelial mass were detected. The addition of caffeine (10 mmol l(-1)) at the beginning of incubation increased PL activity and decreased mycelial mass, while caffeine added after 12 and 24 h resulted in decreases in PL activity and increases in mycelial mass. The results presented here indicate that methylxanthines, especially caffeine, can induce PL in P. griseoroseum.

The best conditions for the production and regeneration of Penicillium expansum and P. griseoroseum protoplasts were the following: 0.5 M KCl, pH 5.5, as an osmotic stabilizer; Novozym 234 at 1.7 mg/ml for P. expansum and 3.3 or 6.7 mg/ml for P. griseoroseum; I to 2 hours of digestion at 25 degrees C; shaking at 130 rpm for I-hour digestion times and 100 rpm for longer periods. These conditions were defined based on the results of protoplast production and regeneration, and resulted in a protoplast yield in the range of 10(7) protoplasts/ml. For protoplast regeneration, 0.56 M sucrose was selected as an osmotic stabilizer for the regeneration medium with an incubation temperature of 25 degrees C. Under these conditions, maximum percentages of protoplast regeneration, based on rite means for all experiments, were 46.5% for P. expansum and 56.9% for P. griseoroseum.

Humicola sp. was cultured on potato-dextrose-agar (PDA) with the pH adjusted to 7.2 with K2HPO4 or KOH, or not (pH 5.76). The cultures were compared for their sporulation capacity in the dark and in the presence of tungsten or fluorescent lamps (6 volts/5 W and 15 volts/54 W, respectively) at 40 degrees C (fungal growth) and 20 degrees C.The best sporulation was obtained in the medium with the pH adjusted with K2HPO4 and incubated under exposure to fluorescent light at 20 degrees C.

In order to obtain pectinase from hyperproducing strains, Penicillium expansum was mutated with UV and NTG. The selected mutants were analyzed for pectinolytic enzyme production and compared with the parental line. Mutants with up to a twofold increase in pectin lyase or polygalacturonase activity were obtained. Mutant P462 presented a two-fold increase in activity of both enzymes.

Polygalacturonase activity of Penicillium expansum was evaluated in the culture filtrate after removal of the mycelial mass. The best culture condition for enzyme production was growth in unbuffered mineral medium in the absence of yeast extract. Production of the enzyme was dependent on growth medium pH and was maximal at acid pH.

AbstractPolygalacturonase (PG) synthesis by the fungus Penicillium expansum in the presence of a combination of pectin and sucrose corresponding to 0.3% of the substrate was repressed more intensely by increasing sucrose concentrations and decreasing pectin concentrations. PG synthesis was partially repressed when sucrose was added 6 or 12 h after the beginning of culture and fully repressed when sucrose was added at the beginning of culture. Tests with actidione indicated that repression by sucrose occurs at the transcription level. We suggest that an accumulation of messenger RNA occurred when the fungus was cultured for 36 h in 0.3% pectin and actidione since, after being washed and reincubated in actidione‐free medium in the presence of the repressor, the fungus synthesized PG up to 54 h of culture, when a reduction in accumulated messenger RNA and PG synthesis occurred. Repression by sucrose was reversible since the fungus started synthesizing PG again in the absence of the repressor and in the presence of the inducer.

Sucrose, a non-pectic carbon source, with yeast extract (YE) added was able to support the production of pectin lyase (PL) by Penicillium griseoroseum Dierckx. However, in the absence of YE, the fungus did not produce PL but grew and caused a marked reduction in culture medium pH. Furthermore, in the absence of YE, only a culture medium with a high buffering capacity permitted the production of PL in the presence of pectin. On the other hand, in the presence of 0.06% YE and of 0.1% pectin, the fungus produced maximum growth and specific PL activity during a 48-h period of culture, with a small variation in medium pH. In the absence of sucrose, YE concentrations from 0 to 0.16% did not support enzyme production, indicating synergism between sucrose and YE for production of the enzyme.

Penicillium griseoroseum Dierckx produced pectin lyase, when grown in liquid culture containing citrus pectin as the sole carbon source.Temperature, pH, buffers and calcium ion for the action of pectin lyase (PL) from P. griseoroserum were studied. PL cleaved citrus pectin most efficiently at pH 6.0 and 50-degrees-C. Sorensen's phosphate buffer allowed the greatest enzymatic activity, and calcium ions did not stimulate enzyme activity. PL exhibited activity on ramie fibres and citrus pectin but did not cleave polygalacturonic acid.

Partial characterization of Enterobacter sp. phosphotransacetylase points to pH and temperature optima in the range of 7.0 to 8.0 and close to 40-degrees-C, respectively. The enzyme is very labile at elevated temperatures and stable in the pH values ranging from 5.0 to 9.0. The Km values at 25-degrees-C, pH 7.4 for CoA and acetyl-phosphate are 1.77 x 10(-4) M and 3.94 x 10(-3) M, respectively. Activation energy amounts to 21 kj mol-1. Phosphotransacetylase is stabilized by the joint action of ammonium sulphate and dithioerythritol. It exhibits remarkable similarity in terms of kinetic parameter values to phosphotransacetylase from some other bacteria.

The yeast Candida guilliermondii produced approximately 10.0-mu-g/ml of riboflavin in a synthetic medium containing xylose as a source of carbon and energy. The excretion of riboflavin was favoured in pH 7.0, in xylose concentration up to 6.0%, and by a 50 rpm agitation. A supplementation of the fermentation medium with cobalt chloride (10(-5) M), dimethyl sulfoxide (1 M) and asparagine (1.5 g/l) exerced an activating effect on the flavinogenesis.

Two strains of Kluyveromyces fragilis (145 and 276) and one of Kluyveromyces lactis were tested for their abilities to produce beta-D-galactosidase in cheese whey. Kluyveromyces fragilis 145 was selected for its higher beta-D-galactosidase activity per cell at the end of the exponential growth phase.