Microcosm experiments.

Microcosms were used to conduct in situ studies on the survival of E. coli in beach sand. The microcosm design was adapted from the work of Alm et al. (52). Microcosms were constructed from pieces of polyvinyl chloride pipe 9 cm long and 5 cm in diameter and were sealed with polyvinyl chloride knockout test caps that had approximately 30 1-mm-diameter holes drilled through them. The end caps were lined with two 0.22-µm filters on the inner sides, which prevented bacteria and other microbes from entering or leaving the microcosms while allowing for oxygen and moisture to enter. The microcosms were filled with various sand treatments, seeded with E. coli, sealed with silicone sealant, and buried 0.5 m deep in the backshore sand of Lake Michigan in Milwaukee, WI, USA. The native sand treatment mimicked the conditions at the beach and consisted of sand collected directly from the beach, with its native microbial community and available nutrient content intact. The autoclaved treatment consisted of moist sand collected from the beach that was autoclaved for 1 h, which inactivated the native microbial community, releasing nutrients from biological constituents and making organic nutrients highly available (Table 1). Last, the baked sand treatment created a nutrient-limited environment; it consisted of sand that was baked in a muffle furnace for 3 h at 550°C, then washed with sterile Milli-Q water, and autoclaved to sterilize it. Isolates used in the experiments were grown for 18 h in MUG medium, washed three times with DNA-free sterile water, and then diluted to a final concentration of 10⁶ cells/ml by using optical density (OD) values.

The source microcosm experiment entailed pooling populations of 176 isolates (each) from gull waste, sewage, and beach sand by source and then inoculating the pooled isolates into native and autoclaved sand. The nutrient microcosm experiment entailed inoculation with the beach sand isolate collection. Native sand microcosms were buried for 45 days, and autoclaved microcosms were buried for 96 days, with an additional set of microcosms left for 320 and 360 days to test long-term survivability. The following summer season, a nutrient microcosm experiment was conducted in which the same population of beach isolates (n = 176) was seeded into native, autoclaved, and baked sand, and microcosms were buried at the beach for 56 days. Included in this experiment were two nutrient dose treatments, in which native and baked sand were seeded with 10% (by weight) autoclaved sand, which acted as a nutrient source. The nutrient-spiked treatment was compared to the nonspiked treatment in order to determine the effects of competition on E. coli survival in beach sand. Isolates from the end of each experiment were recovered for phylotyping and were stored in 96-well microtiter plates in a 25% glycerol–75% MUG medium solution in a −80°C freezer.