Dr1900

COD, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen were determined using the reagent rapid determination methods (HACH DR1900, Loveland, CO, USA). The total nitrogen concentration was regarded as the sum of all nitrogen species. Dissolved oxygen (DO), pH, and oxidation-reduction potential (ORP) were monitored by a multiparameter (WTW3420, Munich, Germany). The fixed biomass concentration of the bio-carrier was calculated as VSS (g/L) based on the dry weight method [27 (link)].

Physico-chemical analyses were conducted weekly for the influent and effluent of the different HFCWs. Chemical oxygen demand (COD), NH₄-N, and NO₃-N were measured with Hach Lange test cells (LCK 400, 304, and 349, respectively, Düsseldorf, Germany) on a spectrophotometer (DR 1900, Hach Lange/Dortmund, Germany). Electrolytic conductivity (EC) and pH were determined by using a multi-parameter water quality meter (SensoDirect 150, Loviband/Dortmund, Germany).

Biomass from eight sites in YNP (Table S1A) were collected and processed as previously described (11 (link)). Briefly, samples were collected in 2017 using sterilized forceps or pliers and stored on dry ice in transit. DNA (250 mg) was extracted using the Qiagen Powersoil kit following the manufacturer’s protocol. Sulfide, Fe²⁺, and dissolved silica were measured onsite using a DR1900 portable spectrophotometer (Hach Company, Loveland, CO). Water samples were filtered through 0.2-μm polyethersulfone syringe filters (VWR International, Radnor, PA, USA) and analyzed for dissolved inorganic carbon (DIC) concentration, δ¹³C and δ¹⁵N as described previously (25 (link)). Field blanks composed of filtered 18.2 MΩ/cm deionized water, transported to the field in 1-L Nalgene bottles, were processed on site using the equipment and techniques previously described (11 (link)). To determine site dissimilarity, we generated a principal-component analysis using sample water geochemistry, geographic location, and biofilm isotopic data (Table S1A) (11 (link)). We converted all raw data to Z-scores (z = x – mean(x)/sd(x)), and principal components of transformed data were generated using the rda function in vegan (69 ) and plotted using ggplot2.

For the determination of the content of chlorophyll a + b (mg·100 g⁻¹ fresh weight, FW) and carotenoids (µg·100 g⁻¹ FW) [14 (link),15 (link),83 (link)], the freshly collected celeriac leaves (0.4 g) were comminuted to a smooth paste in a mortar with a few drops of acetone (80%), pinch of sand and calcium carbonate. Then, the mixture was filtered using Schott filter and vacuum pump, quantitatively transferred to a volumetric flask (50 mL) and filled up with the solvent. The measurements (in four replicates) of absorbance (663, 645, and 470 nm) were made immediately after preparing the solutions with the use of portable visible spectrophotometer (HACH DR1900, Berlin, Germany). The formulas for the calculations are presented in our previous study [14 (link),15 (link)].
The survey of the greenness index (in 10 replicates) of leaf blades was performed using SPAD 502 Plus Chlorophyll Meter (Konica Minolta, Osaka, Japan).
The colour of leaves (in 10 replicates) was assessed using MiniScan (Hunter Lab EZ, Reston, VA, USA). The L value for each scale indicates the level of light (numbers from 51 to 100) or dark (numbers from 0 to 50), the a value indicates redness (positive number) or greenness (negative number), and the b value yellowness (positive number) or blueness (negative number). All these three values are required to completely describe leaves’ colour.

Samples for dissolved organic carbon (DOC), nitrite, nitrate and ammonium measurements were filtered at 0.45 μm (Nanocolor Chromafil membrane filter GF/PET 0.45 μm, Macherey Nagel, Düren, Germany) for sample conservation and stored at 4°C before the chemical analysis. DOC was measured using a total organic carbon analyzer (Shimadzu TOC-L, Kyoto, Japan). Ammonium was measured by gas-diffusion flow injection (Foss, Hillerød, Demark). Nitrite and nitrate were measured by means of ion chromatography (Metrohm 881, Herisau, Switzerland).
Free and total chlorine were measured immediately after sampling using a portable spectrophotometer (DR 1900, Hach, Loveland, USA) with corresponding test kits (DPD, 0–2 mg/L free chlorine, Hach, Loveland, USA).

The contents of chlorophyll a + b and carotenoids were determined in fresh leaves. Samples (0.4 g) were disintegrated, using a mortar and a pestle with the addition of a few drops of acetone (80%), a pinch of sand, and calcium carbonate. The obtained mixture was filtered, transferred to a volumetric flask (50 ml) and filled with acetone. The absorbances were measured in four replicates at 663, 645, and 470 nm with the use of a spectrophotometer (HACH DR1900, Berlin, Germany). The greenness indexes of the leaves were evaluated, using an SPAD 502 Plus Chlorophyll Meter (Konica Minolta, Osaka, Japan), and the colors of the leaves were measured, using MiniScan (Hunter Lab EZ, Reston, Virginia, USA) (in 10 replicates).