Isolation of novel benzo[a]anthracene-degrading microorganisms and continuous bioremediation in an expanded-bed bioreactor.

In the present work, several samples from lab waste containers polluted with polycyclic aromatic hydrocarbons (PAHs) and heavy metals were investigated as potential sources of PAH-degrading microorganisms. After isolating, two fungal strains were selected as the best degrading microorganisms. Genetic identification by sequencing was carried out and they were identified as Trichoderma longibrachiatum and Byssochlamys spectabilis. Their degradation ability was determined in liquid cultures with 100 µM of benzo[a]anthracene. T. longibrachiatum cultures showed highest degradation values (around 97%) after 9 days, furthermore in a second batch the time was reduced to 6 days. To analyse the viability of industrial application, a continuous treatment in an expanded-bed bioreactor was developed operating at different residence times with T. longibrachiatum immobilised on cubes of nylon sponge. It is noticeable that the bioreactor working in continuous mode was able to operate without operational problems and attaining high degradation levels depending on the residence time.

Paecilomyces variotii xylanase production, purification and characterization with antioxidant xylo-oligosaccharides production.

Paecilomyces variotii xylanase was, produced in stirred tank bioreactor with yield of 760 U/mL and purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.29-fold purification with 34.47% activity recovery. The enzyme purity was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirming its monomeric nature as single band at 32 KDa. Zymography showed xylan hydrolysis activity at the same band. The purified enzyme had optimum activity at 60 °C and pH 5.0. The pH stability range was 5-9 and the temperature stability was up 70 °C. Fe2+and Fe3+ exhibited inhibition of xylanase enzyme while Cu2+, Ca2+, Mg2+ and Mn2+ stimulated its activity. Mercaptoethanol stimulated its activity; however, Na2-EDTA and SDS inhibited its activity. The purified xylanase could hydrolyze beechwood xylan but not carboxymethyl cellulose (CMC), avicel or soluble starch. Paecilomyces variotii xylanase Km and Vmax for beechwood were determined to be 3.33 mg/mL and 5555 U/mg, respectively. The produced xylanase enzyme applied on beech xylan resulted in different types of XOS. The antioxidant activity of xylo-oligosaccharides increased from 15.22 to 70.57% when the extract concentration was increased from 0.1 to 1.5 mg/mL. The enzyme characteristics and kinetic parameters indicated its high efficiency in the hydrolysis of xylan and its potential effectiveness in lignocellulosic hydrolysis and other industrial application. It also suggests the potential of xylanase enzyme for production of XOS from biomass which are useful in food and pharmaceutical industries.

Modelling the effect of temperature and water activity on the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea.

The purpose of the present study was to apply a modelling approach to define the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea on a synthetic medium as a function of temperature and water activity. Both fungal species were grown on malt extract agar at different temperatures (10, 15, 20, 25, 30, 35, 40 and 45 degrees C) and a(w) levels (0.88, 0.90, 0.92, 0.94, 0.96 and 0.99) for a period of 30 days. Growth responses were evaluated over time in terms of colony diameter changes. Growth data were fitted to the primary model of Baranyi and the resulting growth rates were further modeled as a function of temperature and water activity using the cardinal model with inflection (CMI) (Rosso et al., 1993). A logistic regression quadratic polynomial model was also employed to predict the probability of growth over storage time. Estimated parameters for minimum, maximum and optimum temperatures for growth were 9.1 degrees C, 46.4 degrees C and 32.1 degrees C for B. fulva and 10.5 degrees C, 43.2 degrees C and 32.1 degrees C for B. nivea. The respective values for a(w) were 0.893, 0.993 and 0.985 for B. fulva and 0.892, 0.992 and 0.984 for B. nivea. No growth was observed at 0.88 a(w) regardless of temperature for both species, whereas B. nivea ascospores could not grow at 10 and 45 degrees C irrespective of a(w). Regarding growth boundaries, the degree of agreement between predictions and observations was >98% concordant for both species. The erroneously predicted growth cases were 1.4-4.2% false positive and 2.1-3.5% false negative for B. nivea and B. fulva, respectively. The developed logistic model was validated with two literature data sets as well as with data from independent experiments carried out on fruit juices. Validation results showed that agreement with literature data for growth was 25 out of 36 (69.4%) cases, whereas validation on fruit juice data failed in only 6 cases (5 false positives and 1 false negative) out of 128 cases.

Depletion of pentachlorophenol in soil microcosms with Byssochlamys nivea and Scopulariopsis brumptii as detoxification agents.

Pentachlorophenol (PCP) is a toxic compound which is widely used as a wood preservative product and general biocide. It is persistent in the environment and has been classified as a persistent organic pollutant to be reclaimed in many countries. Fungal bioremediation is an emerging approach to rehabilitating areas fouled by recalcitrant xenobiotics. In the present study, we isolated two fungal strains from an artificially PCP-contaminated soil during a long-term bioremediation study and evaluated their potential as bioremediation agents in depletion and detoxification of PCP in soil microcosms. The two fungal strains were identified as: Byssochlamys nivea (Westling, 1909) and Scopulariopsis brumptii (Salvanet-Duval, 1935). PCP removal and toxicity were examined during 28 days of incubation. Bioaugmented microcosms revealed a 60% and 62% PCP removal by B. nivea and S. brumptii, respectively. Co-inoculation of B. nivea and S. brumptii showed a synergetic effect on PCP removal resulting in 95% and 80% PCP decrease when initial concentrations were 12.5 and 25 mg kg-1, respectively. Detoxification in bioaugmented soil and the efficient role of fungi in the rehabilitation of PCP contaminated soil were experimentally proven by toxicity assays. A decrease in inhibition of bioluminescence of Escherichia coli HB101 pUCD607 and an increase of germination index of mustard (Brassica alba) seeds were observed in the decontaminated soils.

Novel nonadride, heptadride and maleic acid metabolites from the byssochlamic acid producer Byssochlamys fulva IMI 40021 - an insight into the biosynthesis of maleidrides.

The filamentous fungus Byssochlamys fulva strain IMI 40021 produces (+)-byssochlamic acid 1, its novel dihydroanalogue 2 and four related secondary metabolites. Agnestadrides A, 17 and B, 18 constitute a novel class of seven-membered ring, maleic anhydride-containing (hence termed heptadride) natural products. The putative maleic anhydride precursor 5 for both nonadride and heptadride biosynthesis was isolated as a fermentation product for the first time and its structure confirmed by synthesis. Acid 5 undergoes facile decarboxylation to anhydride 6. The generic term maleidrides is proposed to encompass biosynthetically-related compounds containing maleic anhydride moieties fused to an alicyclic ring, varying in size and substituents.

Reducing patulin contamination in apple juice by using inactive yeast.

The mycotoxin, patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one), is a secondary metabolite produced mainly in rotten parts of fruits and vegetables, most notably apples and apple products, by a wide range of fungal species in the genera Penicillium, Aspergillus, and Byssochlamys. Due to its mutagenic and teratogenic nature and possible health risks to consumers, many countries have regulations to reduce levels of patulin in apple products. In the present study, reduction of patulin contamination in apple juice by using 10 different inactivated yeast strains was assessed. Our results indicated that nearly twofold differences in biomass existed among the 10 yeast strains. Eight of the 10 inactivated yeast strains could provide >50% patulin reduction in apple juice within 24 h, with the highest reduction rate being >72%. Furthermore, juice quality parameters, i.e., degrees Brix, total sugar, titratable acidity, color value, and clarity, of the treated apple juice were very similar to those of the untreated patulin-free juice. Potential applications of using inactivated yeast strain for patulin control are also discussed.

Use of a logistic model to assess spoilage by Byssochlamys fulva in clarified apple juice.

The percentage P (%) of spoiled bottles (n=40) of clarified apple juice due to Byssochlamys fulva, was modeled by using a logistic model: P = P(max)/1 + exp (k(tau-t)) where P(max) (%) the maximum percentage of spoiled bottles, k (h(-1)) a slope parameter and tau (h) the time for P=P(max)/2. Bottles of pasteurized apple juice were inoculated with B. fulva IOC 4518 ascospores for low and high initial loads, 4.8+/-2.3 ascospores/100mL and 19.3+/-4.6 ascospores/100mL respectively and incubated at 21 degrees C and 30 degrees C. P(max) was not significantly different from 100% except for a low initial load at 21 degrees C. Model parameters were estimated with a good accuracy, RMSE in the range 3.89-7.50. Then the model was used to determine the time for 10% bottles spoiled, t(10%). This time was greater at low initial loads, 57.4 and 104 h at 30 and 21 degrees C respectively, than at high initial loads 23.9 and 75.1h at 30 and 21 degrees C respectively. This study demonstrated that even at a very low initial contamination, clarified apple juice can be easily spoiled by B. fulva highlighting the importance of controlling critical control steps of fruit juice processing (i.e., fruit washing, juice filtration and pasteurization).

Influence of package, type of apple juice and temperature on the production of patulin by Byssochlamys nivea and Byssochlamys fulva.

Although the production of patulin in apple fruits is mainly by Penicillium expansum, there is no information on the ability of heat resistant moulds that may survive pasteurization to produce this mycotoxin in juice packages during storage and distribution. In this study, the production of patulin by Byssochlamys spp (Byssochlamys nivea FRR 4421, B. nivea ATCC 24008 and Byssochlamys fulva IOC 4518) in cloudy and clarified apple juices packaged in laminated paperboard packages or in polyethylene terephthalate bottles (PET) and stored at both 21 degrees C and 30 degrees C, was investigated. The three Byssochlamys strains were able to produce patulin in both cloudy and clarified apple juices. Overall, the lower the storage temperature, the lower the patulin levels and mycelium dry weight in the apple juices (p<0.05). The greatest variations in pH and degrees Brix were observed in the juices from which the greatest mycelium dry weights were recovered. The maximum levels of patulin recovered from the juices were ca. 150 microg/kg at 21 degrees C and 220 microg/kg at 30 degrees C. HPLC-UV, HPCL-DAD and mass spectrometry analyses confirmed the ability of B. fulva IOC 4518 to produce patulin. Due to the heat resistance of B. nivea and B. fulva and their ability to produce patulin either in PET bottles or in laminated paperboard packages, the control of contamination and the incidence of these fungi should be a matter of concern for food safety. Control measures taken by juice industries must also focus on controlling the ascospores of heat resistant moulds.