Dataset: Metabolite Uptake Incubations - Microbial Abundances (Flow Cytometry)

Three metabolite uptake incubation experiments were conducted in January 2021 based on the results from previous incubations. These experiments were designed to assess if reef seawater microbial communities assimilate specific exometabolites released by corals and if the microbial communities respond distinctly to different metabolites. Experiments were conducted with three metabolites in separate incubations occurring on different days: riboflavin, pantothenic acid, and caffeine. Riboflavin and pantothenic acid were chosen because both metabolites were released by at least 4/6 species within the organism incubation experiments and because both are B vitamins. Caffeine was selected because it was released in high quantities by R. textilis. Samples were collected at three time points over the course of the incubation: 0, 6, and 24 hrs. Additionally, incubations were conducted in the dark and processed in low-light conditions to minimize photodegradation of the metabolites, especially riboflavin.

Before the incubations were conducted, purified metabolite standards were diluted with MQ water to 5 nM (riboflavin, D-pantothenic acid) and 10 nM (caffeine) one month prior to the incubations, frozen, and shipped to the USVI. Metabolite dilutions were prepared in combusted amber vials and kept in the dark to minimize photochemical degradation. The incubations were conducted in St. John, U. S. Virgin Islands from January 16th-January 21st, 2021.

To set up the incubations, 5.5 l of seawater was collected approximately 1 m above the reef at Tektite reef (9 m depth) using Niskin bottles deployed by divers. Half of the seawater was filtered using a 0.1 µm, 47 mm Omnipore filter to create filtered seawater (FSW), while the rest of the seawater was filtered using a 1 µm, 47 mm Omnipore filter in order to remove larger phytoplankton and protistan grazers, but retain the picoplanktonic microbial community. The filtrate treatments were poured into 36 acid-washed and autoclaved 125 ml polycarbonate bottles. Due to the small volumes and to minimize the chance of sample contamination, the incubations were designed so that bottles could be sampled by sacrifice at each of the three timepoints. Each incubation had four different experimental conditions: 3 different controls and one experimental treatment. The three controls included only FSW (F), FSW with the addition of a metabolite spike (F + Mtb), and 1 µm filtered seawater containing microbes (M). The experimental condition was 1 µm filtered seawater containing microbes with the addition of the metabolite (M + Mtb). Metabolite spikes (20 pM for riboflavin and pantothenic acid and 70 pM for caffeine) were added to the FSW + Mtb and M + Mtb treatments by removing 500 µl (riboflavin and pantothenic acid incubations) or 875 µl (caffeine incubation) of seawater from each incubation bottle, replacing the lost volume with the appropriate metabolite spike, and inverting the bottles to mix. After the metabolite spikes were added, 24 of the 36 incubation bottles were placed in a flow-through seawater table covered with a tarp and equipped with a HOBO data logger to monitor relative light levels and water temperature, while the remaining 12 bottles, accounting for triplicate bottles of each of the four experimental conditions, were immediately processed for the initial timepoint.

After each time point, samples from the incubation bottles were directly collected for assessment of microbial biomass via flow cytometry or re-filtered using 0.2 µm, 47 mm Omnipore filters. The filters were placed into cryovials, frozen in a dry shipper, and the filtrate was collected.