Phytoplankton biomass was estimated using chlorophyll a by collecting ~125 ml of lake water in West Thumb of Yellowstone Lake (fig. S1) from 5-m depth in 2004, 1.2 liters of water from 5-, 10-, and 15-m depths in 2005 (18 ), and 1.2 liters of water from 5- and 15-m depths during 2016–2017. Chlorophyll a was collected on 7 dates (n = 56), 3 dates (n = 36), 11 dates (n = 88), and 9 dates (n = 72) in 2004, 2005, 2016, and 2017, respectively. To concentrate phytoplankton, water was filtered through 25-mm PALL type A/E glass fiber filters. Chlorophyll a was extracted by incubating filters in 90% ethanol buffered with MgCO3 overnight, and concentration was measured using the acid method with a pheopigment correction (58 ) on a fluorometer (Turner Designs 700, Sunnyvale, CA). A secondary solid standard was calibrated using a primary chlorophyll a commercial standard of Anacystis nidulans (Sigma-Aldrich). Chlorophyll a before lake trout invasion was measured to 20-m depth in West Thumb during 1972 by acetone extraction and a spectrophotometer (19 ). Differences in extraction methods were corrected for (58 ).
Anacystis nidulans
Manufactured by Merck Group
Anacystis nidulans is a cyanobacterium commonly used as a model organism in laboratory settings. It is a single-celled, photosynthetic prokaryote that can be cultivated in various growth media. Anacystis nidulans is frequently utilized for studies related to photosynthesis, cell biology, and molecular genetics.
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4 protocols using anacystis nidulans
1
Phytoplankton Biomass Estimation in Yellowstone Lake
2
Measuring Diatom Biomass via Chlorophyll a
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Chlorophyll (chl) a was measured as a proxy for diatom biomass according to EPA Method 445 [54 ]. Both SSW and AGG samples were filtered onto 0.7 μm GF/F and stored at -20°C. Pigment was extracted overnight in the dark at 4°C with a 40:60 dimethyl sulfoxide: 90% acetone solution and then measured using a benchtop 10AU Turner Designs fluorometer. A chlorophyll standard extracted from the alga Anacystis nidulans (Sigma—Aldrich) was used to prepare a standard curve.
3
Quantifying Microalgal Biomass via Chl a
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Microalgal biomass was quantified by chlorophyll a (chl a) fluorescence, using a Turner Designs Trilogy Fluorometer. Chl a was extracted in 90% acetone at 4 °C for 24 h (Parsons et al., 1984) . A chl a standard (Anacystis nidulans -Sigma) was used to calibrate the fluorometer.
4
Assessment of Pelagic Ecosystem Dynamics
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Temperature data, at the time of mussel collection, was obtained from a Sea Bird Electronic, SBE 19 Plus CTD probe. Surface water samples were collected at the surface from the Niskin bottle at the end of the trophic state of different systems. After careful mixing, subsamples were distributed to determine inorganic nutrient and phytoplankton biomass (in terms of chlorophyll-a).
To determine nutrient concentrations (NO3-, NO2-, NH4+, Si(OH)4, PO43-) samples were collected in 20 mL low-density polyethylene containers and stored at 20°C until further analysis. The analyses were performed using a Flow Sys System autoanalyzer, following the procedure described by Hansen and Grasshoff (1983) . 500 mL of seawater were filtered into 25 mm GF/F Whatman filters for phytoplankton biomass. Filters were stored in liquid nitrogen until further analysis. The analyses of chlorophyll-a (Chl-a) and phaeopigments (Phaeo) were carried out according to Jeffrey and Humphrey (1975) (link), with a spectrofluorometer (Spex), which was checked daily with a Chl-a standard solution (from Anacystis nidulans; Sigma).
To determine nutrient concentrations (NO3-, NO2-, NH4+, Si(OH)4, PO43-) samples were collected in 20 mL low-density polyethylene containers and stored at 20°C until further analysis. The analyses were performed using a Flow Sys System autoanalyzer, following the procedure described by Hansen and Grasshoff (1983) . 500 mL of seawater were filtered into 25 mm GF/F Whatman filters for phytoplankton biomass. Filters were stored in liquid nitrogen until further analysis. The analyses of chlorophyll-a (Chl-a) and phaeopigments (Phaeo) were carried out according to Jeffrey and Humphrey (1975) (link), with a spectrofluorometer (Spex), which was checked daily with a Chl-a standard solution (from Anacystis nidulans; Sigma).
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