Seven compact

To follow the development of the pH of the medium during cultivation, samples were taken from the outlet of the reactor and measured with a pH meter (Seven Compact, Mettler Toledo, Switzerland). As a reference, the pH of the fresh medium was determined. Samples were taken in duplicates.

For various heavy metals measurements, the cadmium (Cd), chromium (Cr), lead (Pb), arsenic (As), copper (Cu), nickel (Ni) and cobalt (Co) were determined in this study. To measure the concentration of metals, 0.5 g soils from each sample were digested with a mixture of concentrated HCl–HNO₃–HF–HClO₄ on a hot plate^{15 (link),28 (link)}. First, a soil sample (0.5 g) was weighed and digested with 5 ml HCl on an electric hot plate at 190 °C until the solution was reduced to 3 ml. Second, approximately 5 ml HNO₃, 5 ml HF, and 3 ml HClO₄ were added to the solution and were digested until no black material remained. After cooling, the digestion solution was moved to a volumetric flask and was diluted to 50 ml. Finally, the digestion solution was filtered through a Millipore filter (0.45 μm), and then the concentration of heavy metals was analyzed by using inductively coupled mass spectrometry (ICP-MS, NexION 300X, Perkin Elmer, Melville, NY, USA). The quality control (reference material: GSB07-3272-2015 (ESS-5) and blank samples were generated. The recovery rates of Cd, Cr, Pb, As, Cu, Ni and Co in the reference material were 92%, 93%, 104%, 96%, 92%, 89% and 86%, respectively. The soil pH was also analyzed using a pH meter (Seven Compact, Mettler–Toledo, Greifensee, Switzerland) in 2.5:1 water/soil suspensions in this study.

The pH of different concentrations of the dye extract (CM) of the calyces of H. sabdariffa and the pH stability were determined and followed for 1 month using a digital pH meter (Seven Compact, Mettler Toledo, USA). The pH of the samples was read and recorded at the beginning of every week until the 4th week.

Contaminated soil was collected from the surface horizon (0–20cm) of a paddy field near a smelter (26°15′28.49″N and 118°15′12.78″E) in Youxi County, central Fujian Province, China. The soil samples were air-dried, ground, passed through a 2-mm sieve, and then analyzed (Table 4). Soil pH was determined using a pH meter (SevenCompact; Mettler-Toledo, Greifensee, Switzerland) in 5:1 water/soil suspensions. Cation-exchange capacity (CEC) was determined using 1 M ammonium acetate buffered at pH 7.0. Soil organic matter was determined using the K₂Cr₂O₇ wet oxidation method. Soil particle-size distribution (<2 mm fractions) was determined using the pipette method after hydrogen peroxide treatments33 .

Total soluble solid contents (Brix) were carried out at 20°C using an Abbe refractometer (Bellingham Stanley Limit 60/70 Refractometer, UK). pH was obtained by directly measuring fruit‐based water kefir samples using a pH meter (Seven Compact, Mettler Toledo, USA) equipped with a glass electrode. pH measurement was done in duplicate, and the experiment was conducted in triplicate during fermentation.

All water samples were collected in Northern Portugal on the 6th of May, 2017, between 15 and 16 h. High tide was at 13.13 h and low tide at 19.16 h; however, a limited influence of seawater was expected as the samples were collected in the first 1 m of the water column. River water sample was collected from Lima river in Viana do Castelo (41°41′17.7′’ N 8°47′23.9′’ W), lake water sample from lake of São Pedro de Arcos in Ponte de Lima (41°45′52.5′’ N 8°38′13.60′’ W), and estuary water from Lima river near the mouth of the river (41°40′58.14′’ N 8°49′35.67′’ W). pH was measured using a benchtop pHmeter (SevenCompact, Mettler Toledo) with an accuracy of ± 0.002. All other physical-chemical parameters of water were measured using the Sera Aqua-Test (Table S1).

A sample ≥ 40 µL of the fluid and mucus mixture was obtained after 48 h treatment. To minimize the effect of the diffusion of carbonic dioxide, present in the cell incubator and plausibly in equilibrium with the fluid, ASL samples were rapidly collected in the incubator, and immediately measured (within 2 min) using a microelectrode (SevenCompact, Mettler-Toledo, Novate Milanese, Italy). The measurements were repeated at least in triplicate, discarding the discordant measurements.