Isolation and Identification of Cellulytic Fungi from Agrowastes and Sawmill Soils.

Five agricultural wastes and sawmill soil were investigated for fungi capable of producing cellulase. Isolation was carried using serial dilution techniques and isolates were screened for cellulase production on carboxymethyl cellulose-containing agar plates. Copious isolates were further subjected to submerged cultivation for quantitative evaluation of cellulase biosynthesis. Highest fungal load was 6.67±0.33×106 sfu/g (Sawmill soil) and the least was 2.33±0.33×106 sfu/g (yam peels). A total of sixty seven fungi were identified from the samples. The isolates were of fifteen fungal species namely Mucor mucedo, Aspergillus niger, A. repens, A. flavus, A. parasiticus, Articulospora inflata, Gonatobotrys simplex, Gyrothrix circinata, Dendrospora erecta, Penicillium notatum, P. italicum, Varicosporium elodea, Gonatobotryum apicolatum, Mucor racemosus and Rhizopus nigricans. Aspergillus had the highest occurrence (36.11%), while the least occurrence was Varicosporium and Gonatobotryum (2.78%). Highest cellulase activity ratio was exhibited by Aspergillus niger (1.90) and the least by Aspergillus repens (1.04) on plate screening. The highest cellulase producer among selected isolates under submerged growth was Gonatobotrys simplex (1.2143±0.02 U/ml), followed by Aspergillus niger (1.1429±0.01 U/ml) and the least by Aspergillus parasiticus (0.8265±0.01 U/ml). Submerged protein content was highest in Aspergillus niger culture and least in Penicillium notatum. The study has revealed array of cellulytic fungi from agricultural wastes and sawmill soil.

Produção de proteases por fungos filamentosos isolados do cerrado do centro-oeste brasileiro / Protease production by filamentous fungi isolated from the midwestern Brazilian Cerrado

Proteases ácidas pertencem a um importante grupo de enzimas industriais produzidas por fungos filamentosos, com aplicações na indústria de alimentos, de couro, farmacêutica e de cosméticos. O objetivo principal deste trabalho foi avaliar a produção de proteases ácidas extracelulares de fungos filamentosos isolados do solo do cerrado do centro-oeste brasileiro. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 linhagens de fungos quanto à produção de protease em meio de cultura contendo Agar-leite. O fungo Aspergillus foetidus foi selecionado como melhor produtor de protease ácida extracelular. Visando à otimização da produção de proteases pelo fungo selecionado, avaliou-se a influência de diversos fatores no cultivo (pH, temperatura, agitação e diferentes fontes de nitrogênio e carbono). Após essa etapa, um planejamento experimental estatístico foi realizado com as variáveis independentes temperatura, pH inicial do meio e fonte de carbono e nitrogênio. A produção máxima de protease foi encontrada (63,7 U/mL) nas condições: pH inicial do meio igual a 7,0 a 28 ºC, 150 rpm em peptona 2% (p/v). Os estudos em biorreator demonstraram produção de protease nas condições de agitação e aeração iguais à 300 rpm e 1,0 vvm, após 120 h de cultivo. Os ensaios com diferentes temperaturas para a estimativa dos parâmetros termodinâmicos demonstraram que a protease ácida produzida pelo fungo é altamente estável apresentando máxima atividade em pH 5,0 e temperatura ótima igual a 55ºC. E, finalmente, para a purificação da enzima foi realizada cromatografia de gel-filtração. A enzima apresentou massa molecular de 50,6 kDa, e a análise do zimograma confirmou a atividade proteolítica. Além disso, a protease purificada foi inibida pelo composto pepstatina, indicando uma característica de protease ácida. Esses resultados obtidos demonstram um fungo filamentoso produtor de uma nova protease ácida com potencial aplicação para indústria farmacêutica e de cosméticos

The acid proteases belong to the most important group of industrial enzymes produced by filamentous fungi, with applications in the food, leather, pharmaceutical and cosmetics industries. This study aimed the evaluation of extracellular acid proteases production from filamentous fungi isolated from different samples of the midwestern Brazil cerrado. Initially, a screening was performed to assess the ability of the 17 strains of yeast for production of protease-agar medium containing milk culture. The Aspergillus foetidus was selected as the best producer. Aimed at optimizing the production of proteases by the selected fungus, first evaluated the influence of various factors on the cultivation (pH, temperatura, agitation and different sources of nitrogen and carbon). After this step, a statistical experimental design was carried out with the independent variables temperatura, initial pH of the medium and source of carbon and nitrogen. The best conditions for protease production were (63.7 U / mL): initial pH values greater than 7.0, at 28 °C, 150 rpm peptone 2% (w/v). Aiming future production of this protease in industrial scale, studies have shown better in bioreactor protease production under the conditions of agitation and aeration equal to 300 rpm and 1.0 vvm, after 120 h of cultive. The tests at different temperaturas to estimate the thermodynamic parameters showed that the acid protease produced by the fungus is highly stable with maximum activity at pH 5.0 and optimum temperatura of 55 °C. And finally, for the purification of the enzyme were performed gel-filtration chromatography. The enzyme had a molecular mass of 50.6 kDa, and the analysis of the zymogram showed a proteolytic band. Furthermore, the purified protease was inhibited by pepstatin compound, indicating a feature of acid protease. These results demonstrate a new filamentous fungus producing acid protease with potential application to pharmaceuticals and cosmetics

Growth of the Ectomycorrhizal Fungus Pisolithus Microcarpus in different nutritional conditions

The most important plant species employed in reforestation programs depend on ectomycorrhizal fungi for their establishment and growth. The exploitation of this symbiosis to improve forest productivity requires fungal inoculants in a large scale level. To develop such a technology it is necessary to define the optimal composition of the culture medium for each fungus. With these objectives in mind, the effect of the composition of the culture medium on biomass production of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) was studied. The original composition of two culture media, already employed for cultivation of ectomycorrhizal fungi, was submitted to several variations with the C/N ratio as the main variable. A variation of the Pridham-Gottlieb medium was the most efficient for the production of biomass. Therefore, it was submitted to a factorial assay where glucose, peptone and yeast extract components were the factors analyzed. Results showed that the glucose concentration may be increased up to 40 % in order to promote higher biomass production. Peptone had a positive effect on this variable, whereas yeast extract promoted a deleterious effect. These results indicate that it is advisable to eliminate yeast extract from the medium and replace it with peptone prior to use.

Growth of the Ectomycorrhizal Fungus Pisolithus Microcarpus in different nutritional conditions

The most important plant species employed in reforestation programs depend on ectomycorrhizal fungi for their establishment and growth. The exploitation of this symbiosis to improve forest productivity requires fungal inoculants in a large scale level. To develop such a technology it is necessary to define the optimal composition of the culture medium for each fungus. With these objectives in mind, the effect of the composition of the culture medium on biomass production of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) was studied. The original composition of two culture media, already employed for cultivation of ectomycorrhizal fungi, was submitted to several variations with the C/N ratio as the main variable. A variation of the Pridham-Gottlieb medium was the most efficient for the production of biomass. Therefore, it was submitted to a factorial assay where glucose, peptone and yeast extract components were the factors analyzed. Results showed that the glucose concentration may be increased up to 40 percent in order to promote higher biomass production. Peptone had a positive effect on this variable, whereas yeast extract promoted a deleterious effect. These results indicate that it is advisable to eliminate yeast extract from the medium and replace it with peptone prior to use.

Production of endoglucanase by the native strains of Streptomyces isolates in submerged fermentation

Cellulase is a complex enzyme system, commercially produced by filamentous fungi under solid-state and submerged cultivation. It has wide applicability in textile, food and beverage industry for effective saccharification process. In this study, cellulolytic enzyme activity, particularly endoglucanase of 26 Streptomyces strains isolated from garden soil was examined, including two isolates selected on the basis of potential cellulolytic activity on Bennett's agar medium. To enhance the endoglucanase formation in broth culture, different conditions including carbon and nitrogen sources, and growth conditions were tested. The maximum endoglucanase activity (11.25-11.90 U/mL) was achieved within 72-88 h in fermentation medium containing Tween-80, followed by phosphate sources. Both cellulolytic Streptomyces isolates gave almost equal quantity of enzyme in all trials. However the effect of medium ingredients on endoglucanase induction diverged with strains in some extent.

A celulase é um sistema enzimático complexo, produzido comercialmente a partir de fungos filamentosos através de cultivo em estádio sólido e submerso. Tem uma grande aplicação na indústria têxtil e de alimentos e bebidas no processo de sacarificação. Nesse estudo, examinou-se a atividade celulolítica, especialmente de englucanase, de 26 cepas de Streptomyces isoladas de solo, incluindo duas cepas selecionadas por sua atividade celulolítica no ágar Bennett. Para estimular a produção de englucanase em meio de cultura, diferentes condições de cultivo, incluindo fonte de carbono e nitrogênio e condições de crescimento, foram avaliadas. A atividade máxima de glucanase (11,25 a 11,90 U/mL) foi obtida em 72-88h em meio de cultura contendo Tween-80, seguido por fontes de fosfato. Ambas as cepas celulolíticas de Streptomyces produziram quase a mesma quantidade de enzima em todos os experimentos. Entretanto, o efeito dos ingredientes do meio na indução da glucanase divergiu de acordo com a cepa.