Structure and Activity of a Microbial Community Treating Acid Mine Drainage

Sage R. Hiibel1, Amy Pruden2, and Kenneth F. Reardon1. (1) Chemical and Biological Engineering, Colorado State University, 100 Glover Building, 1370 Campus Delivery, Fort Collins, CO 80523-1370, (2) Civil and Environmental Engineering, Colorado State University, Engineering Research Center, 1320 Campus Delivery, Fort Collins, CO 80523-1320

Acid mine drainage poses a significant problem to the environment and public health, impacting more than 20,000 km of streams and waterways in the United States alone. AMD is typically high in sulfates and heavy metals, and low in pH. Biological treatment, through the implementation of a sulfate-reducing permeable reactive zone (SR-PRZ) that is installed transverse to flow, is an attractive remediation option because of the low cost and maintenance requirements. SR-PRZs contain organic substrates, usually solid, that support the growth of a complex microbial community, including sulfate-reducing bacteria (SRB). The SRB form H2S which then reacts with heavy metals to precipitate stable metal sulfides. To date, the microbial aspect of SR-PRZs has typically been treated as a black box. Since SR-PRZ operation is highly dependent on microbial activity, a better understanding of the role of the mixed microbial culture in these systems is needed. This research uses a suite of molecular biological tools to profile the structure and activity of the microbial communities responsible for AMD treatment. Cloning and denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA from two sets of columns remediating AMD has shown that those microorganisms capable of degrading cellulose (and other polysaccharides) and fermenting the products of the hydrolysis represent the majority of the microbial community. SRB bacteria, known to represent a relatively low proportion of the microbial community in other sulfate-reducing mine drainage treatment systems, were only detected in the columns most effectively treating AMD using DGGE. Further analysis with quantitative real-time PCR (Q-PCR) confirmed the presence of SRB in all columns (below the DGGE detection limit). Interestingly, sequences related to Bacteroides spp., known for their ability to utilize a wide variety of compounds as carbon and energy sources, were also only detected in the set of columns most effectively treating AMD. The DGGE and cloning results were confirmed using capillary electrophoresis single-strand conformational polymorphism (CE-SSCP). CE-SSCP is an extremely sensitive, high throughput molecular technique that uses electrophoretic mobility to separate the single-strand conformation of nucleic acid sequences. When coupled with the 16S rDNA clone library already developed, CE-SSCP eliminates the need for cloning and sequencing to identify the species present in subsequent samples. CE-SSCP analysis has also been performed on the 16S rRNA of the microbial communities to provide insight into which community members are actively involved in the remediation of AMD. The cellular ratio of total RNA to DNA has been shown to be proportional to growth rate in a variety of organisms, and thus can be utilized to determine which members of a mixed culture are actively growing and which microorganisms are dormant. The relative amounts of each can also be tracked over time to determine how the activities of community members change in response to environmental factors. This analysis is expected to illuminate the specific members of the overall community actively involved in AMD remediation. A better understanding of the active members in microbial communities treating AMD will aid in the development of future SR-PRZs. The ultimate goal of this research is to link microbial activity with remediation performance and to design ideal microbial inocula for AMD treatment.