Research Article
Ewa Beata Górska, Urszu
Abstract
The bioconversion of cellulose to soluble sugars by diazotrophic bacteria is a very important for the environment, such as for the global stabilization and a sustainable human society. Two nitrogen-fixing microorganisms hydrolyzing cellulose were isolated from agricultural soil and identified as Paenibacillus polymyxa [the laboratory names EG2 and EG14] based on 16Sr RNA sequence. The genome of these bacteria was found to carry nif genes coding the individual components of the nitrogenase complex. Their nitrogen fixing ability was confirmed by studying nitrogenase activity in cultures of the studied bacteria in N-free medium supplemented with carboxymethylcellulose (CMC). The nitrogenase activity of P. polymyxa EG 2 was 2.9 nM C2H4?ml-1?h-1 whereas P. polymyxa EG 14 0.4nM C2H4?ml-1?h-1. The isolates in medium with filter paper synthesize following cellulolytic enzymes: carboxymethylcellulase (CMCase), FPase and Avicellase. Of the cellulolytic enzymes in the culture supernatants of the bacteria the most abundant was CMCase (P. polymyxa EG 2 103.4 mU, EG 14:96.1 mU) with far lower amounts of enzymes hydrolyzing crystalline Avicel cellulose or filter paper. In spite of these observations the better isolate in terms of synthesis of cellulases is P. polymyxa EG 14. Zymograms reflecting the main cellulase activities of the studied bacteria do not significantly differ from each other and present at least three major enzymatic activities with high molecular masses: one of about 200 kDa, another of about 220 kDa and a strong band of activity with mass of about 130 KDa. Observations of the bacterial cultures in medium with filter paper revealed the colonization of the substrate by single cells or aggregates of bacterial cells surrounded by slime. Scanning and transmission microscopy of the isolates revealed the presence of spherical structures resembling cellulosomes on the surface of the bacteria being characteristic for anaerobic bacteria of the genus Clostridium.