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- Characterization of Cultivated Fungi Isolated from Grape Marc Wastes Through the Use of Amplified rDNA Restriction Analysis and Sequencing
- Spyridon Ntougias , Nektarios Kavroulakis , Kalliope K. Papadopoulou , Constantinos Ehaliotis , Georgios I. Zervakis
- J. Microbiol. 2010;48(3):297-306. Published online June 23, 2010
- DOI: https://doi.org/10.1007/s12275-010-9193-y
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- 8 Scopus
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Abstract
- Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7×106 per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.
Research Support, Non-U.S. Gov't
- Purification and Biochemical Properties of a Glucose-Stimulated β-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse
- Cesar Vanderlei Nascimento , Flávio Henrique Moreira Souza , Douglas Chodi Masui , Francisco Assis Leone , Rosane Marina Peralta , João Atílio Jorge , Rosa Prazeres Melo Furriel
- J. Microbiol. 2010;48(1):53-62. Published online March 11, 2010
- DOI: https://doi.org/10.1007/s12275-009-0159-x
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- 57 Scopus
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Abstract
- The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-galactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.
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