Characterizing the Abundance and Activity of Soil Microbes by Capillary Electrophoresis Using Single-Strand Conformational Polymorphism

Alice C. Jernigan1, Greg Thoma1, Duane C. Wolf2, and Christa N. Hestekin1. (1) Chemical Engineering, University of Arkansas, 3202 Bell Engineering, Fayetteville, AR 72701, (2) Crop, Soil and Enviornmental Sciences, University of Arkansas, 3202 Bell Engineering, Fayetteville, AR 72701

Bioremediation is the process by which microbes are used to break down soil contaminates. Although bioremediation has the potential to be a useful and potentially less expensive and more natural method of soil reclamation, there is limited information about the actual microbial communities that perform the remediation and how their abundance and activity changes with time. The identification and assessment of activity of microbial soil communities can be accomplished by isolating DNA and RNA from soil samples. These DNA/RNA samples can then be characterized by a technique called single-strand conformational polymorphism (SSCP) analysis of the 16S gene. The 16S gene is highly conserved among microorganisms, but has unique small variable regions that can be used to differentiate between species. These regions can be amplified by polymerase chain reaction (PCR) giving small fragments of the gene sequence. SSCP, a method that is normally used to detect mutations, is the heat denaturing of these fragments resulting in single strand DNA. The refolding of the single strand DNA will form different secondary structures (conformations) depending on the nucleotide sequence. These different conformations can be separated due to their different electrophoretic mobility, even if they are the same size. The traditional method for looking at bioremediation is by viable plate counts or microscopic examination. These methods are both time consuming and can underestimate the numbers due to the inability to culture many soil organisms. The increased speed and automation of capillary electrophoresis (CE)-SSCP will allow for more rapid and reliable results. By isolating both DNA and RNA from the same soil sample, at the same time, a more accurate picture of the microbial community in those samples will be available. This will be important in the study of contaminated soils and the monitoring the progress of bioremediation.