Production of Cellulases by the Endophytic Fungus Fusarium oxysporum

Cellulose has enormous potential as a renewable energy source and the application of cellu lases in the conversion of cellulo lytic biomass may provide great economical benefits; in addition, cellulases can be used in a wide range of industrial applications. Given the b iotechnological importance of cellulases, the aim of this study was to evaluate the production of cellulases by endophytic fungi o f the genus Fusarium oxysporum isolated from Baccharis dracunculifolia D.C. (Asteraceae). The studies were conducted using a basic substrate of sugarcane bagasse that was pretreated for complete removal of the sugar content. The material was dried at 28C for 96 days and quantified every seven days after 25 days of fermentation. To quantify the enzymes, the indirect spectrophotometric method was used, with DNS reagent. The results showed that the greatest peak of enzyme production was at 55 days of fermentation, with a yield of 55.21 ± 10.54 IU/g of fermented substrate, at pH 5.96. Thus, it can be concluded that the fungus Fusarium oxysporum is a producer of cellu lase enzymes.


Introduction
The term cellu losic or lignocellulosic material is used to describe the major constituents of most plants, i.e., cellulose, hemicellulose and lignin, the co mposition of which not only depends on the type of plant, but also on growth conditions [1].
Cellu lose is the most abundant organic polymer on Earth and the main component of plant biomass. It is found in pure form, as in cotton, but is often found associated with hemicellulose and lignin in the cell wall [2]. Cellulose has a relatively simple structure, comprising of D-glucose mono mers linked by glycosidic bonds β-1, 4.
Hydrolase enzy mes are widely used in various industries. Cellu lases and amilases are enzy mes fro m the most economically important group of carbohydrolases. The high consumption of cellu lases may be explained by their wide use in the food industry, in the process of extract ion of vegetable oils, in the maceration of fru it and clarification of juices, as well as in the manufacture of beer and wine; they are also used in the formulation of an imal feeds [3].
Filamentous fungi are the most widely used type of fungi in industry for the production of cellulases, especially those of the genera Aspergillus, Trichoderma, Humicola, Penicillium, Fusarium and Phanerochaete [4]. Currently, cellu lases are the third most industrially produced enzymes world wide because of their applications, for example in cotton processing, paper recycling, the extract ion of juices, as enzymatic detergents and as animal food additives [2,5].
The identification of the activ ity of en zy mes in microorganis ms and the studies related to optimizing their production are of great industrial interest. The isolation of samples with d ifferent production potentials of en zy mes may represent alternatives for industry. Furthermore, the isolation of fungi fro m natural environ ments may result in lineages with greater production potential and that are more adapted to the environment [6].
Biotechnological processes have gained a prominent place in worldwide technological develop ment, and have economic and operational features that have mo re advantages than conventional chemical p rocesses [7]. Annually, 5.4 x 10 8 tons of cane sugar are processed in the world; on average, one ton of sugar cane generates 280 kg of bagasse [8].
Approximately 50% of the bagasse is used in power generation in power plants, while the remainder is stored. Given the importance and the increased production of sugar cane bagasse as industrial waste, there is growing interest in developing methods of producing biofuels and chemical products that offer economic and environ mental advantages.
Many trials have been carried out to degrade this complex material into simple, fermentable sugars for ethanol production. The degradation of cellulose by cellulases is a very well studied process and the role of these enzy mes is affected by porosity and crystallin ity of cellu lose as well as the lignin and hemicellulose content of bio mass [9]. Endophytic microorganisms were first discussed at the beginning of the nineteenth century, but the first person who distinguished between them and plant pathogens was Bary in 1866. Generally, all plants have endophytic microorganisms. A single plant may contain several, including fungi and bacteria. There may be quite frequent species in a part icular host, which are called the dominant species, in contrast to other more rare ones, which are called the secondary species [10].
According to Martin [11], the endophytic fungi of the genus Fusarium are distributed worldwide and are found in the soil or associated with species of plants; they decompose organic matter in the soil and can cause many diseases in different plant species.
The plant species Baccharis dracunculifolia D.C. (Asteraceae) has biological and ecological importance for the isolation of epiphytic and endophytic microorganisms, due to its adaptability to the diverse biomes of the Americas, particularly South America. It is represented by more than 500 species distributed mainly in Brazil, Argentina, Colo mb ia, Ch ile and Mexico, and occupies the higher regions.
The high concentration of species in Brazil and the Andes indicates that one of these areas is the probable center of origin of this genus. They are usually shrubs, and in Brazil are co mmonly called vassoura or vassourinha (which translates as broom, little broo m) and measure 0.5 to 4.0 meters in height [12].

Microorganisms
This study used the strain D3-FB of the endophytic fungus

Determination of Cellulolytic Acti vity
The cellulo lytic act ivity was measured using a basic support of sugarcane bagasse that had been rinsed successively in running water for complete removal of sugars. The washed bagasse was dried in a fan oven at 65 °C for 24 hours, and then was packed in polyethylene bags and stored in dry conditions.

Fermentati on i n Erlenmeyer Fl asks
Cellobiose (1%) and carbo xy methylcellulose (1%) were added to the sugar cane bagasse substrate to induce the production of cellu lases and also as initial carbon sources of the med iu m. This mixture was inoculated with a suspension of 5 g of the fungus inoculums, wh ich had been previously grown on a rice culture. It was then homogenized in an Erlen meyer flask and incubated at 28 °C for 69 days.

Anal ysis of Fermented Substrate
Aliquots of five grams of the med iu m were collected every 7 days and mixed with 50 ml of d istilled water in the presence of a 7.0 buffer. This suspension was continuously agitated for 30 minutes. It was then filtered to remove solids to yield a clear ext ract used for pH determination. The extract was centrifuged at 3000 rp m for 15 minutes and the supernatant was considered as an enzyme source to determine the reducing sugars via the indirect spectrophotometric method. The indirect spectrophotometric method was used to determine enzy me activity, based on the release of glucose molecules by the action of cellulolyt ic enzy me co mplex.

pH
The pH was measured on a suspension obtained after homogenizat ion of 5 grams of ferment in 50 mL o f distilled water, which was continuously agitated for 30 minutes.

Measurement of Reducing Sugars
The reducing sugars were measured by the reaction with 3,5-din itrosalicy lic "DNS" [13]. In an alkaline mediu m and at elevated temperature, 3,5-din itrosalicylic turns into 3-amino-5-nit rosalicylic. It acquires a yellowish coffee color that absorbs at 540 n m. One unit of cellulases was defined as the amount of released enzyme capable of acting on the substrate and releasing 1 μmo l of reducing sugar (exp ressed as glucose) per minute.

Statistical Analysis
The statistical analysis was carried out using the program Statistica, version 5.0. Analyses of variance were in line with ANOVA standards. The significant differences between means were determined using the Tukey's test. All act ivities were triplicated.

Results and Discussion
The data concerning the behavior of the endophytic fungus Fusarium oxysporum are shown in Fig 1. of fermentation, respectively. The data show that the period of greatest yield of the enzy matic co mplex was at 55 days of fermentation with a production of 55.21 ± 10.54 IU/g, at pH 5.96. Figure 1 shows that at the start of the fermentation process, when there is availability of cellobiose and carboxy methylc ellu lose, these supplements acted as inducers for the metabolism o f the fungus Fusarium oxysporum. They therefore init iated the production of enzymatic co mp lex, which reacted with the cellu lolytic co mponents, thus degrading the cellulose and releasing glucose. The data obtained in this study are similar to those obtained by Paschoalatti [14], who studied the in vivo and in vitro production of cellulase enzymes by the fungi Sclerotinia sp., Rhizoctonia solani, Fusarium sp., Penicillium sp. and Pythium ultimum; this similarity suggests that these enzymes play an important role, since cellu lose is the major co mponent of plant cell walls and all of them were ab le to degrade cellu lose.
The results obtained in this study, are in line with Braga et al. [15], who reported that the fungus Fusarium sp. grew in med iu m containing cellulose as the sole carbon source: evidence of its ability to synthesize cellulolytic en zy mes. All of the 33 strains tested were capable of degrading the cellu lose-based substrate in semisolid fermentation based on sugar cane bagasse.
Shihata, Abdou and Galal [16] assessed the production of cellu lase enzyme, and reported that F. moniliforme, F. oxysporum and F. solani, isolated from pea, are producers of pectinase and cellulase enzy mes both in vitro and in vivo. In the study by Bueno et al. [17], it was observed that the isolates of F. solani of the passion fru it plant, produce a greater variety of extracellular enzy mes, including amylases, lipases, cellu lases, proteases, laccases and catalases and that the amounts of enzymes produced were different in each of the isolates.
This behavior justifies what Aguiar and Menezes [18] reported in a study evaluating the total cellulase activity obtained with sugar cane bagasse inoculated with Aspergillus niger: they found a yield of 25 IU/gram of fermented substrate after 168 hours fermentation. Thus, the data fro m different fermentation conditions show that the fungus Aspergillus niger has the ability to secrete higher quantities of cellulase than the fungus Fusarium oxysporum, which was used in this study, in a shorter period of time.
It is important to highlight that the species of microorganis m and the temperature are important variables, as they may d irectly interfere in the production of en zy mes. In this study, the temperature was maintained at 28°C.
The monitoring of pH is also important, because according to Soccol [13], the fungus has a capacity for growth, albeit limited, under ext reme conditions of acidity and alkalin ity. These features are ext remely important for fermentation processes, because they show that under these conditions the vast majority of the bacteria responsible for the contamination of the fermentation processes are inhibited.

Conclusions
Fro m the present study, one can conclude that the fungus Fusarium oxysporum is a producer of cellulolytic en zy mes, and may thus be used in biotechnological processes to obtain these enzymes or to produce glucose. The sugarcane bagasse was effective at inducing Fusarium oxysporum to produce the cellulase enzy mes. The highest level of en zy me production was observed at 55 days of fermentation at a constant temperature of 28℃, with an enzy matic production of 55.21 ± 10.54 IU/g of fermented substrate, at pH 5.96.