MOL2NET , 2017 , 3, doi:10.3390/mol2net-03-xxxx 2 showing 0.0073 - PDF document

MOL2NET , 2017 , 3, doi:10.3390/mol2net-03-xxxx 1 MOL2NET, International Conference Series on Multidisciplinary Sciences MDPI http://sciforum.net/conference/mol2net-03 Obtaining microorganisms with cellulolytic activity in different regions of Ecuador Carmen A. Salvador Pinos (pochasalvador@yahoo.com) a , Maira Rojas Carrillo (maira.rojas@udla.edu.ec) b , Leyanis Mesa Garriga (leyanis.mesa@gmail.com) c , Pablo Londoño (palondono@uce.edu.ec) a , Jonathan Villavicencio Montoya (jfvm_ 1993@hotmail.com) c , Erenio González Suárez (erenio@uclv.edu.cu) c a Universidad Central del Ecuador b Universidad de las Américas c Universidad Central Marta Abreu de las Villas Abstract. It is estimated that in developing countries 60% of waste is from lignocellulosic material that can be used in recycling processes. The purpose of this research is to find enzymatic cocktails from bacteria native to Ecuador that allow degradation of the lignocellulosic material, by searching native microorganisms collected in Los Andes, Amazonia and Antartida, which can be introduced in a process of bagasse enzymatic hydrolysis on an industrial scale. For this, qualitative and quantitative tests were carried out to measure the endoglucanase, exoglucanase and filter paper activity of the microorganisms and their enzymatic cocktails. Enzymatic hydrolysis tests were also performed on sugarcane bagasse. In addition, the value of the investments for the production of enzymatic cocktail of bacteria Bacillus sp . and the total cost of production was calculated. It was identified that Peniccillium sp . was the species with the highest activity of filter paper,

MOL2NET , 2017 , 3, doi:10.3390/mol2net-03-xxxx 2 showing 0.0073 UFP and a glucose yield of 11,96 in 100 grams of bagasse, which opens the possibility of its use, in industrial processes. From this best microorganism strain, the equipment was sized on an industrial scale and it was concluded that the investment cost would benefit the country. The study shows that it is possible to generate a suitable technology for the production of cellulolytic enzyme crudes in Ecuador. Keywords: Cellulases, Bacillus, enzymatic cocktail, bagasse, sugar cane Introduction Microorganisms play an important role in the carbon cycle as well as cellulolytic enzymes are essential for the degradation of the cell walls of plants. However, there are limitations to understanding the functioning of this process: the vast diversity of microorganisms in the environment and the complexity to cultivate them (Gomashe et al., 2013). The most studied microorganisms for industrial purposes are fungi and bacteria, because generally, they are groups that present extracellular enzymes secreted in the medium in which they are cultivated (Gobat Jean & Willy, 1998). Enzymes are biological catalysts that come from different environments, but most of them are of microbial origin (Prabha et al., 2007). There are two types of enzymes: extracellular and intracellular. The extracellular or exoenzymes, are synthesized inside the cell, forming a coctkail or crude enzyme that are subsequently secreted by the metabolism that allows the cell to make exchanges with the environment in which it lives. The intracellular ones are synthesized and used inside the cell or they are linked to subcellular particles or intracellular membranes, and their extraction is more difficult. To elucidate the mechanism in which they act is complex, since they do it through the contact of the substrate with the enzyme through the aminoacids of the active site. To collect these enzymatic cocktails, releasing processes are carried out, such as cell lysis for the extraction or collection of the enzymatic crude from the medium in which the microorganism is found (Gobat, 1998). Vegetable biomass is the most abundant renewable resource on the planet; this is an attractive source for obtaining bioenergy and biochemical products. It is composed of lignin, cellulose and hemicellulose (Gonzalo de Gonzalo, 2016). The raw material, in which these studies are carried out, does not compete with the food biomass. The largest fraction of organic matter in terrestrial ecosystems is made up of cellulose. Soil microorganisms are the main responsible for transferring carbon from cellulose and other compounds to the atmosphere, but their contribution is not exactly known (Hairchar, 2007). One of the agro- industrial products with a large amount of cellulose is the sugarcane bagasse, which is a residue of the

MOL2NET , 2017 , 3, doi:10.3390/mol2net-03-xxxx 3 stems after juice extraction, which is generally used in sugar factories as boiler fuel (Pandey et al. ., 2000). Due to the potential of biomass as a renewable source for the production of fuels and chemical products, with the modern metagenomic techniques, several studies have been carried out to understand the cellulose-degrading microorganisms. However, the mechanism of degradation of cellulose is still not clear and what microbial communities carry out this work. Those are important reasons for increasing research in these areas (Wilson, 2011). In addition to reducing the volume of agro-industrial or urban lignocellulosic waste by 40%, the cellulose-degrading microorganisms diminish the problems of treatment, management and risk of urban, agricultural and forestry waste. The objective of the study was to find microorganisms native to Ecuador capable of producing cellulolytic enzymes that can be used on an industrial scale for bagasse degradation and to perform the analysis of the cost of investment and total production. Materials and Methods (optional), no page limit Collection of lignocellulosic material Remnants of lignocellulosic material were supplied from an artisan panela 1 factory in Balzapamba (foothills of Los Andes), in the reforestation program of Petroproduction (Amazonia) and around Pedro Vicente Maldonado Station (Antartida). The samples collected in the aforementioned sites were washed with distilled water, in the same way as for the study of microbiota in other species (Lauer et al., 2007). The microbiota was then analyzed using techniques that are commonly used for the analysis of cellulolytic microorganisms (Salvador et al., 2012). For this, 100 grams of lignocellulosic substrate was placed in 1000 ml of peptone water, at 400 rpm, for 20 minutes. The solution with the microorganisms was heated at 80 ° C, for sporulation and were sown in petri dishes. Dilutions of 1:10 were made seven times. Obtaining microorganisms 40 μl of dilutions 4,5 and 6 from the stock solution were sown, they were observed for seventeen days in ACP medium (Agar Count Plate) and PDA (Potatoe Dextrose Agar). The number of colony forming units (CFUs) per cm2 was counted. They were also sown in nutritious broths under the temperature conditions of ± 37˚C for bacteria and at ± 25˚C for fungi until strains growth was observed. The colonies obtained by exhaustion were isolated and purified. Pure strains were stored in cryo tubes at - 80 ° C in the strains collection at the University of the Americas in Quito, Ecuador. Production of cellulase enzyme The isolated sporulating strains were cultured in CMC medium (Carboxymethylcellulose, which is a derivative of cellulose). Petri dishes that had halos of degradation (which are clear zones), were 1 It is a sweetener derived from the sugarcane juice