Molecular phylogenetic characterization of microbial community dynamics associated with freshwater stream environmental organic matter sources

Abstract

The effects of various biotic and abiotic factors on microbial community dynamics during leaf breakdown were assessed through a series of in situ leaf breakdown studies within the Fort Benning Military Installation (FBMI), Georgia, USA. Leaf litter microbial community composition was mainly controlled by incubation time and, to a lesser extent, by leaf chemistry. Instream sediment disturbance and its associated effects on stream physicochemical conditions drastically altered bacterial assemblage composition during leaf breakdown. Chapter 2 described a protocol for purifying genomic DNA from environmental sources for use in a polymerase chain reaction (PCR). This protocol was necessary because many of the techniques utilized in this dissertation involved extraction and amplification of genomic DNA from organic matter and often required purification of the genomic DNA. The protocol involves embedding genomic DNA extract in an agarose plug and incubation within a formamide and saline solution. The purified DNA can then be extracted from the agarose and used as a template for PCR. A test of this protocol using red maple leaf genomic DNA yielded significantly more amplicons using ~20 ng of purified DNA compared to extracted DNA alone. Chapter 3 described a 128-d in situ leaf breakdown study within a single stream at FBMI to assess the effects of shredding macroinvertebrates on leaf litter microbial communities. Contrasting mesh sizes (6.35- and 1-mm mesh) were used to reduce shredder macroinvertebrate abundance, and microbial community composition was characterized over 9 dates. Macroinvertebrate results revealed no reduction in shredder abundance, suggesting that the use of 1-mm mesh may be inappropriate in streams where the dominant shredders are fairly small and slender (e.g., Polypedilum and Leuctra spp.). Chapter 4 described the differences in microbial community composition between leaf species of strongly contrasting leaf chemistries and associated breakdown rates. Maple and oak leaf species were used due to their drastically different leaf chemistries (e.g., higher percent lignin and cellulose in oak). Leaf chemistry differences resulted in significantly different microbial community composition measured using both ribosomal intergenic spacer analysis (RISA) and phospholipid fatty acid analysis (PLFA). These differences in microbial community composition were strongest during early leaf breakdown and decreased over time. Time was the main factor found to structure leaf litter bacterial assemblages with early and later breakdown times having different bacterial assemblage compositions. Chapter 5 described a 64-d in situ leaf breakdown study at FBMI to quantify the effects of sediment disturbance on leaf litter bacterial assemblages. A variety of response variables were measured including several physicochemical conditions (streamwater temperature, pH, depth, current velocity), leaf breakdown, and bacterial assemblage composition (measured using RISA and bar-coded pyrosequencing). The main physicochemical condition measured in this study that affected bacterial assemblage composition of leaf litter was streamwater pH, which was correlated to disturbance intensity, which in turn correlated to sediment. Overall, results showed sediment disturbance significantly altered leaf litter bacterial assemblage composition and was associated with a shift towards an assemblage capable of surviving harsher environmental conditions (e.g., increased pH, decreased dissolved oxygen).