Fungal biodegradation of dibutyl phthalate and toxicity of its breakdown products on the basis of fungal and bacterial growth.

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC50) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC50. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC50 and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation.

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l(-1), with a biomass production of 5.6 g l(-1) and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l(-1), biomass production of 4.5 g l(-1), and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.

Mycelial growth of strains of Pleurotus ostreatus developed on agar and its correlation with the productivity in pilot production farm

Radial growth rate, intracellular laccases and proteases activities, and protein content were evaluated in five strains of Pleurotus ostreatus, grown on starch-based and glucose-based agar media containing different concentrations of the glucose analogue 2-deoxyglucose (2-DG). Productivity of the strains in pilot scale cultivation was also determined. The mycelium of four strains had approximately between 0.6- to 3-fold higher protein content when grown on glucose medium containing 0.01 g/L of 2-DG than when grown on glucose medium. The radial growth rate and intracellular laccases activity of some strains showed a positive and a negative correlation with the productivity, respectively. These results suggest that the strains with high radial growth rate and low intracellular laccases activity on glucose without 2-DG, on starch without 2-DG or on glucose containing 0.01 g/L of 2-DG are highly productive in pilot production farm.

Avaliou-se o crescimento radial, as atividades de proteases e lacases e o conteúdo protéico de cinco cepas de Pleurotus ostreatus cultivado em agar à base de amido e à base de glicose contendo diferentes concentrações de 2-deoxiglicose (2-DG), um análogo da glicose. A produtividade das cepas em cultivo em escala piloto foi também determinada. Em quatro cepas o conteúdo protéico do micélio foi aproximadamente 0,6 a 3 vezes maior quando foram cultivadas em meio à base de glicose contendo 0,01g/L de 2-DG. O crescimento radial e a atividade de lacases apresentaram correlação positiva e negativa, respectivamente, com a produtividade. Esses resultados sugerem que as cepas com elevada taxa de crescimento radial e baixa atividade de lacases em glicose sem 2-DG, ou em amido sem 2-DG, ou em glicose com 0,01 g/L de 2-DG, são altamente produtivas em cultivo em escala piloto.

Growth of Pleurotus ostreatus on wheat straw and wheat-grain-based media: Biochemical aspects and preparation of mushroom inoculum.

Mycelial growth, intracellular activity of proteases, laccases and beta-1,3-glucanases, and cytoplasmic protein were evaluated in the vegetative phase of Pleurotus ostreatus grown on wheat straw and in wheat-grain-based media in Petri dishes and in bottles. The productivity of the wheat straw and wheat-grain-based spawn in cylindrical polyethylene bags containing 5 kg of chopped straw was also determined. We observed high activity of proteases and high content of intracellular protein in cultures grown on wheat straw. This suggests that the proteases are not secreted into the medium and that the protein is an important cellular reserve. On the contrary, cultures grown on wheat straw secreted laccases into the medium, which could be induced by this substrate. P. ostreatus grown on media prepared with a combination of wheat straw and wheat grain showed a high radial growth rate in Petri dishes and a high level of mycelial growth in bottles. The productivities of wheat straw and wheat-grain-based spawn were similar. Our results show that cheaper and more productive mushroom spawn can be prepared by developing the mycelium on wheat straw and wheat-grain-based substrates.

Differential patterns of constitutive intracellular laccases of the vegetative phase of Pleurotus species.

The constitutive intracellular laccase activity of ten strains of Pleurotus spp. was determined in vitro and by zymograms, using different substrates. Differences in the in vitro activities were observed between all the strains; however, zymogram patterns were only similar for strains within same species, independently of any of the three substrate (2,6-dimethoxyphenol, p-anisidine or o-tolidine) used. The differences observed in the number and positions of the isoforms in the gel suggest that laccase zymograms can be used to differentiate species of this organism.

Simple staining detects ultrastructural and biochemical differentiation of vegetative hyphae and fruit body initials in colonies of Pleurotus pulmonarius.

AIMS: To know the ultrastructural and biochemical differences of vegetative hyphae and fruit body initials in colonies of Pleurotus pulmonarius. METHODS AND RESULTS: Feulgen reagent was used to detects differentiation of hyphae. The intracellular laccases, proteases and beta-1,3-glucanases activity, content of cytoplasmic protein, glycogen and glucans in the cell wall were evaluated in hyphae of fruit body initials and in vegetative hyphae. The thickness of hyphal walls of the vegetative hyphae was also evaluated. Substantial biochemical changes were observed in hyphae of different zones of the fruiting colony. Hyphae at the periphery had thinner walls than in the centre of the colony. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Staining correlated with the enzymatic activity, protein, glycogen and glucans, in mycelium and in fruit body initials. The implications are that hyphal maturity in P. pulmonarius involves storage of glucans, in part at least, in the form of a thickened hyphal wall.

Physiology of a colony of Pleurotus pulmonarius grown on medium overlaid with a Cellophane membrane.

The physiology of a colony of Pleurotus pulmonarius grown on potato dextrose agar overlaid with a Cellophane membrane (PDA-WC) was studied. On PDA-WC, the colony presented higher biomass density and productivity of fruit body formation (4.84+/-0.23 mg mycelial biomass/cm2 and 23.7+/-3.12 microg ml(-1) h(-1), respectively) than that which developed on potato dextrose agar without Cellophane (PDA-OC; 0.26+/-0.01 mg mycelial biomass/cm2 and 10.8+/-1.57 microg ml(-1) h(-1), respectively). In cultures developed on PDA-WC, intracellular laccases and beta-1,3-glucanases activities were lower [12+/-0.9 arbitrary units (AU)/g mycelial biomass and 1.33+/-0.1 international units (IU)/g mycelial biomass, respectively] than those observed on PDA-OC (20.65+/-1.0 AU/g mycelial biomass and 3.67+/-0.2 IU/g mycelial biomass, respectively). In cultures developed on PDA-WC, intracellular protein and glycogen concentrations were lower (1.9+/-0.9 and 117+/-3.5 mg/g mycelial biomass, respectively) than those observed on PDA-OC (14.3+/-1.1 and 347+/-2.9 mg/g mycelial biomass, respectively). The radial growth rate and the content of glucans in the cell wall were not significantly different between cultures developed on PDA-WC and PDA-OC. These results show that the use of the Cellophane as a tool to study in vitro fungal physiology might affect the interpretation of experimental results, since the physiology under otherwise similar conditions was different on medium with and without Cellophane.

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study.

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A(w)=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A(w) in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination.