Massimiliano Fenice

Submerged and solid-state production of laccase and Mn-peroxidase by Panus tigrinus on olive mill wastewater-based media.

The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600+/-98 U l(-1) on day 13) and in the air-lift reactor (410+/-22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities.

Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition

This paper reviews current knowledge of the production of organic acids by immobilized microorganisms with a simultaneous solubilization of rock phosphate in fermentation and soil conditions. The most widely applied methods are based on the passive immobilization in preformed porous carriers and entrapment of the microbial cells in natural gels. In general, immobilized systems show higher acid producing and rock phosphate solubilizing activity than freely suspended cells. The potential of gel-entrapped P-solubilizers and mycorrhizal fungi as microbial soil inoculants is also pointed out. Some advantages and constraints of using immobilized cells are discussed and a special emphasis on further research is given.

Production and structural characterization of the exopolysaccharide of the Antarctic fungus Phoma herbarum CCFEE 5080

The filamentous fungus Phoma herbarum CCFEE 5080 isolated from continental Antarctica soil was tested for exopolysaccharide (EPS) production. The fungus grew and produced EPS (up to 13.6 g/l) on a variety of carbon sources among which sorbitol was best, particularly at the concentration of 60 g/l. EPS production was maximum when the nitrogen source was NaNO3 (3 g/l) and the incubation temperature was 28 degrees C. The polysaccharide was purified by repeated precipitation in ethanol and gel filtration and characterized as a homopolymer of glucose having a molecular weight of 7.412 x 10(6); structural analysis indicated the presence of beta-1,3 and beta-1,6 linkages only. After repeated freezing and thawing of the fungal biomass in the presence of EPS, the mycelial growth was much higher than that observed after freezing in the absence of EPS and the difference increased with the number of freeze-thaw cycles. It is hypothesized that the adaptation of P. herbarum CCFEE 5080 to the Antarctic soil microclimatic conditions, characterized by low temperature, high thermal fluctuations and repeated freeze-thaw cycles, might be related to the EPS production ability.

Production of extracellular enzymes by Antarctic fungal strains

Abstractu2002Thirty-three fungal strains, isolated from different sites on Victoria Land (continental Antarctica), were plate-screened for their ability to produce twelve extracellular enzymes. Lipases were generally present and in high quantities in almost all the strains. Polygalacturonase, as well as amylase and phosphatase, was common. Glucose oxidase, protease and DNAase appeared to be generally low or absent. Many strains, producing a limited number of enzymes, appeared to have a low eco-nutritional versatility while a few, such as Verticillium cfr. lecanii no. 1, V. cfr. lecanii no. 3, Aspergillus versicolor and Phoma sp. no. 2, showing a diversified enzymatic competence, are probably advantaged in extreme terrestrial environments characterized by low competition. The possibility of utilizing the enzyme-producing ability of these fungi in applied research is also discussed.

Reuse of microbially treated olive mill wastewater as fertiliser for wheat (Triticum durum Desf.).

Free cells of Aspergillus niger were grown on olive mill wastewater (OMW) supplemented with rock phosphate (RP) in an air-lift bioreactor in batch and repeated-batch processes. The fungus grew well and reduced the chemical oxygen demand of the waste by 35% and 64% in the batch and repeated-batch (fourth batch) processes, respectively. Total sugar content was consistently reduced (ca. 60%) in both processes while reduction of total phenols was minimal. RP was solubilised and maximum soluble P was 0.63 and 0.75 gl(-1) in the batch and repeated-batch (third batch), respectively. Several types of OMW+/-RP, microbially-treated or not, were tested in a greenhouse for their fertilising ability on a soil-wheat (Triticum durum Desf.) model system. Beneficial effects were highest using OMW treated by the repeated-batch process. The treated plants showed an increase in seed biomass, spike number, and kernel weight. Harvest index was highest (0.49+/-0.04) after treatment with OMW from the repeated-batch process.

Rock phosphate solubilization with gluconic acid produced by immobilized Penicillium variabile P16

Penicillium variabile P16 immobilized on polyurethane sponge produced gluconic acid in presence of rock phosphate, the latter being simultaneously solubilized during five repeated batches. A total production of 42, 60, and 90 g gluconic acid/l was obtained for 3, 7, and 14 g rock phosphate/l, respectively. Accordingly, soluble phosphorus concentration increased with gluconic acid production, reaching a maximum of 350 mg/l at the 3d batch in medium supplemented with 14 g rock phosphate/l.

Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79

Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317xa0IU (mg protein)−1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1xa0kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55°C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60°C, the enzyme half-lives were 281 and 25xa0min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99xa0×xa0106 and 3.07xa0×xa0106xa0M−1xa0s−1, respectively). Catalytic rate constants for typical N–OH redox mediators, such as 1-hydroxybenzotriazole (2.6xa0s−1), violuric acid (8.4xa0s−1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8xa0s−1), were found to be higher than those reported for other high redox potential fungal laccases.

Application of encapsulated Penicillium variabile P16 in solubilization of rock phosphate

Abstract Penicicllium variabile P16, encapsulated in agar or calcium alginate, produced gluconic acid in repeated batch shake-flask cultures. Agar-encapsulated mycelium produced 21% more gluconic acid than Ca-alginate-encapsulated fungus, and was utilized in experiments for rock phosphate solubilization. The latter was closely related to gluconic acid production, which was affected by the presence of rock phosphate. Maximum amounts of dissolved P (0.226 gl−1 batch−1) were found at 14.0 gl−1 rock phosphate, but the solubilization efficiency was higher at 3.5 gl−1 rock phosphate. Gluconic acid productivity pattern and the degree of phosphate solubilization were not influenced by decreasing the glucose concentration in the cultivation medium from 80 to 25 gl−1. Encapsulated fungal cells systems could substitute for chemical solubilization of rock phosphate and could be applied as soil microbial inoculants.

Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii

The chitinolytic activity of Verticillium cfr. lecanii A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25 degrees C in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15 degrees C, the levels of enzyme activity of the three strains were similar to those obtained at 25 degrees C. At 5 degrees C, in contrast, the activity of V. lecanii was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h). The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5-60 degrees C): at 5 degrees C, its relative activity was still 50% of that recorded at 40 degrees C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by mycelial damage and cell lysis.

Olive mill waster water treatment by immobilized cells of Aspergillus niger and its enrichment with soluble phosphate

Abstract Olive mill waste water (OMW), supplemented or not with ammonium sulphate and rock phosphate (RP), was applied as a medium in a shake-flask repeated-batch fermentation with Aspergillus niger immobilized on polyurethane sponge. Compared to other treatments, the results showed higher growth of the immobilized mycelium and significant reduction of the total phenols when the waste material was enriched with RP and ammonium sulphate (N). The immobilized fungus solubilized the RP with a maximum level of soluble P of 0·58 g/litre reached during the fourth batch cycle of the OMW+RP treatment. Depending on the medium composition, three types of treated OMW were produced which could be further used for various purposes.