Molecular analysis of bacterial communities associated with a forested wetland impacted by reject coal

Abstract

The Savannah River Site’s D-area harbors an exposed reject coal pile from which acidic, metal-rich saline run-off has impacted an adjacent wetland. Culture-independent 16S rDNA analysis was utilized to assess spatial and temporal bacterial community changes. This environment possesses features unique to acid mine drainage (AMD) systems examined previously. The identification and characterization of the associated bacterial constituents should aid in the understanding of the ecology of AMD systems. The 16S rDNA types recovered via PCR from sites having relatively high and low contamination (RCP1 and RCP2, respectively) were distinct from those recovered from an adjacent unimpacted forested wetland (FW). An interesting trend was the predominant recovery of as yet uncultivated bacteria phylogenetically associated with isolates or environmentally-derived sequences retrieved from habitats having similar chemical characteristics. An exciting discovery was the recovery of several particularly divergent lineages. One of these groups (termed WSRCP) was recovered via a specific PCR assay only from acidic habitats and phylogenetic analysis placed this clade mostly consistently as a novel subdivision of the candidate division WS6. The bacterial communities associated with the RCP1 and RCP2 sites were monitored in five-week increments over a year by both CfoI and MspI T-RFLP analysis and the relative abundance of peak areas between sites were compared. Previously isolated environmental clones related to TRA5-3, TRB3, RCP1-48, as well as Actinomycete, Acidobacterium, and Leptospirillum-related lineages contributed to the predominant peaks. The samples were spatially and temporally differentiated based on principal component analysis of measured environmental parameters and of T-RFLP data. T-RF’s corresponding to Acidobacterium and Actinomycete sequence types were negatively and positively correlated with pH; accordingly these groups were relatively more abundant in the RCP2 and RCP1 samples respectively. In either sample site, TRA5-3, TRB3 and Leptospirillium groups exhibited a strong fluctuation that appeared cyclic in nature. The Leptospirillium group was predominant during the time of year (July-December) when percent moisture values were lower; conversely, the TRA5-3 and TRB3 groups were relatively less abundant in these same samples. In sum, we were able to relate environmental parameters to as yet uncultivated bacteria associated with acid mine drainage-type systems.