APPLICATION OF STABLE ISOTOPE PROBING (DNASIP) IN THE IDENTIFICATION OF CELLULOLYTIC SOIL BACTERIA AND FUNGI
Authors: Feth el Zahar Haichar and Wafa Achouak
Abstract: Cellulose is the most abundant organic polymer on the earth, and represents a huge source of energy for microorganisms. The ability to degrade cellulose is widespread among fungi and bacteria and can take place under aerobic and anaerobic conditions. Microbial degradation of cellulose is characterized by the biosynthesis of multicomponent enzymes, which can be divided into three classes: endoglucanases, exoglucanases and cellobiases. Traditionally, studies of cellulolytic microbes have focused on microorganisms that grow well under laboratory conditions, by using different media containing cellulose from divers origins. However, the major fraction of environmental microorganisms cannot yet be cultured. Recently, a worldwide scientific effort made available several cultivation-independent techniques to classify and understand microbial communities at the molecular level. Among these techniques, stable isotope probing (SIP) approach has been developed to identify functional groups of microbes directly within communities of uncultured microbes. SIP has especially been applied to identify cellulolytic bacteria through the amendment of bacterial 13C- cellulose in soil samples incubated under anaerobic and mesophilic conditions. This approach simultaneously yields information about which microbial populations are present and which members are degrading 13C- cellulose and assimilating derived compounds. Total DNA was extracted from the soil at different incubation periods, and the 13C-enriched DNA from cells that had incorporated 13C derived from labeled cellulose, was separated from unlabelled DNA, corresponding to microbial communities not involved in cellulose degradation, by ultracentrifugation. The identification of active bacterial communities was analyzed and characterized by denaturing gradient gel electrophoresis (DGGE) or by cloning sequencing. Cellulose degradation was associated with significant changes in bacterial community structure. These bacterial populations are closely related to soil bacteria known for their ability to degrade cellulose as well as uncultured bacteria and bacteria not previously known to degrade cellulose in soil. Recently, RNA-stable isotope probing (RNA-SIP) method was also applied in the identification of soil fungi involved in cellulose degradation in forest soil. In conclusion, stable isotope probing provides an important new tool for investigating members of microbial communities that are directly involved in biodegradation of cellulose in soils. The construction of metagenomic libraries from 13C-DNA or 13C-RNA fraction followed by the screening of the clones for cellulase activity will allow us to identify new cellulases having potential applications in industry.