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Molecular Analysis of Bioaugmentation and Biostimulation of Biological Acid Mine Drainage Remediation Systems

Sage R. Hiibel1, Luciana P Pereyra2, Elizabeth Perrault3, 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, 100 Glover Building, 1370 Campus Delivery, Fort Collins, CO 80523-1370, (3) Cell and Molecular Biology, Colorado State University, 100 Glover Building, 1370 Campus Delivery, Fort Collins, CO 80523-1370

The biological treatment of acid mine drainage using sulfate-reducing bioreactors is an an attractive passive, in situ remediation approach. Acid mine drainage is characterized by low pH and elevated levels of sulfates and trace metals. The bioreactors consist of organic substrate, typically wood chips or compost, which supports a complex microbial community. This community includes cellulose degrading and fermentative bacteria that breakdown the cellulosic materials into smaller organic molecules. Sulfate-reducing bacteria utilize the small organics to produce bicarbonate and hydrogen sulfide, which then precipitate the metals in the drainage.

The goal of this work is to investigate the effects of bioaugmentation and biostimulation on sulfate-reducing bioreactors treating acid mine drainage. Anaerobic columns were inoculated with manure from a local dairy farm and fed simulated acid mine drainage. Each column was packed with a an organic substrate mix (beech wood (22 wt%), pine chips (11 wt%), and alfalfa (2 wt%)), limestone (5 wt%), and silica sand (45 wt%). Sulfate and metal removals were determined by ion chromatography and inductively coupled plasma absorbance emission spectroscopy, respectively.

The microbial communities of each column were evaluated at the beginning of the experiment and after the columns had reached steady-state (as determined by sulfate reduction) using a suite of molecular methods. The active members of microbial community were identified using Active Community Profiling, a technique developed in our lab that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) to determine the 16s rRNA to 16s rDNA ratio. Key functional groups of the microbial community were also quantified using quantitative real-time PCR (Q-PCR). Q-PCR targets included family 5 and family 48 glycosyl hydrolases for cellulose degraders , iron only hydrogenases for fermenters, the alpha subunit of the dissimilatory sulfite reductase (dsrA) gene for sulfate reducers, and the alpha subunit of the methyl coenzyme M reductase gene for methanogens.

To explore the effect of bioaugmentation, in addition to the dairy manure one pair of columns were inoculated with cellulose-degrading bacteria and another pair with sulfate-reducing bacteria. Biostimulation effects were investigated by feeding ethanol to one pair of columns and carboxymethyl cellulose to another pair to stimulate sulfate-reducing and cellulose-degrading bacteria, respectively, in addition to the simulated acid mine drainage.

Bioaugmentation of columns with cellulose-degrading bacteria resulted in decreased startup times and increased short- and long-term remediation performance compared to those columns only inoculated with dairy manure. Multiple species identified as cellulose degraders had increased 16s rRNA:rDNA ratios, and the total proportion of cellulose degraders was significantly greater. The columns bioaugmented with sulfate-reducing bacteria also had a decreased startup time and increased short-term remediation performance, but their long-term performance was comparable to the non-augmented columns. The drop in performance was correlated to a decrease in dsrA genes.

The columns supplemented with a readily available cellulose source had elevated proportions of cellulose degraders, fermenters, and sulfate reducers as determined by the glycosyl hydrolases, iron hydrogenases, and dsrA genes, respectively. These columns had a decreased phylogenetic diversity of cellulose degraders and increased levels of short- and long-term remediation performance. Columns supplemented with ethanol followed a similar trend, having elevated proportions but decreased phylogenetic diversity of sulfate reducers, based on dsrA Q-PCR results, and increased short- and long-term remediation performance; the relative proportions of cellulose degraders and fermenters did not change significantly in these columns.

Based on the results of this work, bioaugmentation with cellulose-degrading or sulfate-reducing bacteria does not appear to offer a viable option for long-term remediation performance of bioreactors treating acid mine drainage. Despite decreases in the phylogenetic diversity of the respective functional groups, the biostimulation of cellulose degraders and sulfate reducers gave promising long-term remediation performance results. Additional studies exploring the robustness of the bioaugmented and biostimulated columns with respect to an environmental perturbation are underway.