Iron limitation effects on nitrogen-fixing organisms with implications for cyanobacterial harmful algal blooms. biofilm metagenome

A nutrient manipulation experiment in a facility with 24 stream microcosms located at the University of Texas at Arlington. Briefly, 30.5 cm diameter round glass dishes containing 4.5 L of nutrient media were lined with 36 natural stone tiles (3.1 cm × 1.5 cm × 0.7 cm) for algal colonization. Inoculation of all microcosms took place on day 1 of the experiment by seeding with algae scraped from streams of varying nitrate, phosphate, and iron levels in the Dallas-Fort Worth area. A more thorough description of these microcosms can be found in Larson et al. (2015).Employing a full factorial design resulted in eight different nutrient treatments with three replicates each, hereafter referred to as NPFe, PFe, NFe, NP, N, P, Fe, and Control. However, we focus here only on the NP, NPFe, P, and PFe treatments since biofilms in all other treatments consisted almost entirely of diatoms and did not accumulate sufficient biomass to extract enough DNA for 16S rRNA and nifH genes molecular analyses. On day 60, algal samples from three tiles were randomly chosen from each microcosm and scraped with a toothbrush until visibly cleaned. DNA was extracted and 16S rRNA and nifH genes were amplified and sequenced using 454-sequencing.