| Acid mine drainage (AMD), a common environmental problem caused by mining activities, adversely impacts receiving watersheds, wildlife, and even public health due to its low pH and high concentration of toxic metals. The choice of organic substrates utilized in low-cost, low-maintenance, passive treatment systems for AMD is crucial to maintain a diverse microbial community for successful remediation. Recent studies by our research team have shown that crab shell (CS) amendments improve the longevity and performance of traditional AMD treatment systems containing spent mushroom compost (SMC). However, the microbial contribution to this has not been thoroughly explained. In this research, both chemical and biological techniques were used to comprehensively evaluate microbial communities and their relationship with chemical performance under varying environmental conditions in passive AMD treatment systems amended with CS. This might shed light on future design and operation strategies for AMD bioremediation systems.;Previous performance data of five sulfate-reducing columns treating AMD suggested that columns amended with higher fractions of CS supported more efficient and stable performance in terms of alkalinity generation, sulfate reduction, and metal removal, as compared to columns with traditional SMC and limestone substrates. Accordingly, microbial analyses conducted on packing materials of the five columns in this study revealed significant advantages of CS amendment in sustaining the growth of functionally more diverse microbial groups including cellulose degraders, chitin degraders, fermenters, and sulfur or sulfate reducers. Remarkably, the 100% CS column supported the growth of sulfate-reducing bacteria (SRB) from eight different genera--- key players in AMD treatment systems. PCoA and phylogenetic ARB trees showed that bacterial communities in 50% CS and 100% CS columns were more likely to cluster together. Archaea predominantly identified as methanogens and fungi capable of polysaccharide degradation were only detected in columns containing SMC. In the 100% CS column, copy numbers of the functional genes representing fermenters, sulfate reducers, and chitin degraders were approximately 2.4, 3.9, and 3.2 times higher than those in SMC column, respectively.;In a field study, four pilot-scale vertical flow pond (VFP) systems featuring different substrate combinations of CS and SMC and different underdrain materials were constructed and operated for 633 days at the Klondike-1 site in Cambria County, PA. Analyses of chemical performance data, 454 sequencing, and quantitative PCR (qPCR) data suggested that under changing environmental conditions at the Klondike-1 site, the VFPs containing CS sustained more efficient and reliable treatment of AMD coupled with functionally more diverse and stable microbial communities. Influent pH rapidly increased from < 3.0 to above 6.0 and was maintained circum-neutral by all VFPs. As compared to a control reactor containing SMC, the reactors containing CS sustained higher alkalinity (around 300 mg/L as CaCO3), slower alkalinity exhaustion, and steadier acidity neutralization. Apart from the dissolution of CaCO3 in CS and LS materials, fermentation and sulfate reduction contributed comparably to the generation of alkalinity in pilot VFPs. Correspondingly, copy numbers of genes representing fermenters and sulfate reducers were higher in the pilot reactors containing CS than in the SMC reactor. Similar to the column study, a higher diversity of SRB was observed in reactors containing CS. Due to higher alkalinity levels and sulfate-reducing rates, more thorough metals removal was observed in reactors containing CS than SMC: >90% for the removal of iron (Fe) and aluminum (Al), >50% for manganese (Mn) and zinc (Zn), and no breakthrough above 50% of the influent concentration was observed for Fe, Al, and Zn throughout the course of the test. Geochemical modeling indicated that possible mineral phases of precipitation were goethite and mackinawite for Fe, gibbsite for Al, and rhodochrosite for Mn. Sorption onto the surface of Fe and/or Al minerals could be another mechanism for metal removal. Noticeably, the pilot VFP containing 70% CS was shown to maintain more stable performance while possessing comparable treatment efficiency to the 100% CS reactor over varying environmental and operational conditions during the pilot study. This agrees well with microbial observations under no flow condition, which is experienced by many field bioremediation systems due to seasonal changes or clogging: the relative abundance of core phyla shifted in all pilot reactors, but the smallest changes in functional gene copies were observed in the 70% CS reactor. (Abstract shortened by UMI.). |
