Characterization of cellulolytic bacterial strains from native forest soil from Misiones, Argentina

SABARIS G; GHIO, S; RORIG M; TALIA P; CATALDI A; GRASSO D; CAMPOS E

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General (SAMIGE); 2012

Sociedad Argentina de Microbiología General (SAMIGE)

The use of lignocellulosic biomass for second generation biofuels requires optimization of the biochemical conversion of cellulose to its fermentable components. The objective of this work was the isolation and characterization of cellulolytic bacterial strains from native forest soil in order to identify cellulase encoding genes. A soil sample (0-10 cm) was obtained from a native forest area in Misiones, Argentina. The presence of cellulolytic microorganisms was determined by most probable number assay (MPN). By successive streak out in defined media with carboximethylcellulose (CMC) as sole carbon source, forty-nine colonies were isolated. From these isolates, 37 presented cellulolytic activity visualized by a degradation halo on CMC agar plates stained with Congo Red. Fifteen isolates were selected for further characterization. All of them were able to grow on cristaline cellulose (Avicel) as sole carbon source. Total DNA was obtained from each positive colony and 16s rDNA was amplified with eubacterial specific primers, fD1 and rD1. Amplification products obtained were sequenced and the resulting sequences were compared by Ribosomal Database Project (RDP) and BlastN Genebank NCBI databases. Most isolates corresponded to bacterial consortia, which needed to be maintained in order to keep the cellulolytic activity. Total genomic DNA of the pooled isolates was obtained and used to construct a genomic library. Inserts of 5 kb obtained by partial digestion with Sau3AI were purified and ligated to pBluescript, previously digested with BamHI. Ligation product was used to transform commercial E. coli competent cells. The library was functionally screened for endoglucanase activity on CMC agar plates and Congo Red staining. Positive candidates were further analyzed. This study showed the presence of a diversity of cellulolytic microorganisms in native forest soil from Misiones which indicates that this could be an attractive source for the bioprospection of novel enzymes for cellulose degradation.