Bioprospecção de micro-organismos produtores de celulase de resíduos de sisal

Abstract

In the north-eastern semi-arid region of Brazil, sisal (Agave sisalana) is situated among the main agricultural crops. Sisal’s leaf fibers are used for the manufacturing of rugs, ropes and many different artisanal products. However, only 4% of the whole plant is used, with more than 90% being discharged as residues. These residues are rich in ligno-cellulosic material and constitute a potential source for the isolation of microorganism, as for example enzyme producing organisms. Actinomycetes represents an important portion from soil inhabitant microorganisms as components of the micro-flora in ligno-cellulosic residues as sugar-cane bagasse, sisal’s residues, etc. Due to the high morphological and taxonomical diversity of these organisms, is important to develop prospection studies for industrial and environmental applications. The objective of this work was to select cellulose producing actinomycetes from sisal’s residues and to study enzymatic production of selected strains by means of submerse fermentation, using different substrata as carbon source. Isolates were obtained from Tiquara, Ourolandia, Laje do Batata and Araci counties. These areas were selected due to their production potential. Microorganisms were isolated from 10 g of soil plus 5 g sisal residues in 90 and 95 ml of 0.85% sterile saline solution and diluted until 10-6. Different sterile culture media were used (agar-potato, agar glycerol-peptone and agar amide- casein), where aliquots of 0,1 ml of each dilution were distributed in 3 replicates for each dilution. Pure cultures were obtained from isolated colonies and 166 actinomycetes were selected. In order to isolate cellulolitic producing microorganisms, culture media containing 1,0% carboxi-methylcellulose as the only carbon source was used. Petri dishes were incubated 12 days at 30ºC. Cellulose degradation was observed after adding Red Congo 1% (p/v) solution, subsequently, submerse fermentation was performed in Erlenmeyer flasks using the Central Rotational Compound Design 32, with a total of 11 experiments. From the 166 prospected isolates, 17 were considered as potentially cellulose degradators. However, only one strain, ARA01, was selected for fermentation studies, due to its rapid growth. All fermentations in medium containing sisal residues as well as sugar cane bagasse showed high enzymatic production, although sisal bagasse was the best substratum with 630 U/L versus 260 U/L form sugar-cane bagasse. Statistical analysis showed that milhocin was the Nitrogen source with the highest influence over enzyme production, once the increase on its concentration significantly incremented enzymatic activity, while the increase in carbon sources did not influenced in cellulase activity within the studied range. The preliminary results of this work showed that the isolated microorganism, as well as the substrata used, have potential for cellulase production.