Drb 200

SCP was extracted at the end of the experiment by the alkaline extraction method reported by Perovic et al. with minor modifications [47 (link)]. The 15 mg of lyophilized ground sample was mixed with 10 mL of 0.4 M NaOH and agitated on a compact digital rocker (Model 88880020, Thermo Scientific, USA) at 100 rpm for 30 min. After agitation, samples were sonicated in an ultrasonic bath (CPX1800H-E, Branson, USA) at 70 °C for 2 h followed by digestion at 100 °C for 1 h in a COD digital reactor block (DRB 200, Hach, USA). After samples were cooled down, these samples were centrifuged at a speed of 23,366 g and a temperature of 4 °C, and pellets were discarded. To precipitate the proteins, the pH of the supernatant was adjusted to 3 by adding 1 M HCl, drop by drop. The sample was then centrifuged again, followed by pH adjustment to 12 using 1 M NaOH. The sample was centrifuged a final time and the supernatant was analysed for true protein using the Lowry protein assay method [48 (link)].

The chemical oxygen demand (COD) was determined using a COD colorimeter (DR1010, HACH, Loveland, CO, USA) with a digestion device (DRB200, HACH) according to the US EPA approved HACH Method No. 8000. The pH value was measured using a pH meter (pHS-3C, Leici, INESA Scientific Instrument Co., Ltd., Shanghai, China) with Glass electrode method (Chinese National Standard GB 6920-86). NH₃-N and TP were determined using an ultraviolet/visible spectrophotometer (T6xinyue, HuaBi Scientific Instrument Co., Ltd. Nanjing, China) with Nascar reagent photometry and Chinese National Standard GB 11893-89. PVA was analyzed using the UV–Vis spectrophotometer (DR 6000, HACH) at 690 nm, based on the blue color produced by the reaction of PVA with iodine (analytically pure, Tianjin Zhiyuan Chemical Reagent Co. Ltd., Tianjin, China) in the presence of boric acid (analytically pure, Tianjin Zhiyuan Chemical Reagent Co. Ltd.) [2 (link)]. The turbidity of the PML was determined using a turbidity meter (ET93810, Lovibond^® Tintometer Group, Dortmund, Germany) according to the US EPA 180.1 method. The BOD₅ was determined according to the pressure sensor method with an automated measurement apparatus. The measurement of BOD (20 °C, 5 days) was performed using a pressure sensor method with an automated measurement apparatus (BOD Trak II, HACH).

The water in each system was sampled for water quality at the end of the 2nd, 4th, 6th, and 8th batches. The water temperature, pH, and dissolved oxygen (DO) were measured by a Temperature/Light Data Logger (HOBO UA-002-08; Onset, Cape Cod, MA, USA), a portable Multi-parameter Water Quality Meter (U52; Horiba Ltd., Kyoto, Japan) and a DO electrode (HQ40D-53LED; Hach Company, Loveland, CO, USA), respectively. NH₄⁺–N, NO₂⁻–N, NO₃⁻–N, total nitrogen (TN), and COD were determined through water quality analyzing systems (DRB200 and DR2800; Hach Company, Loveland, CO, USA) according to standard analytical procedures [28 ]. The water sampling was conducted according to guidelines on sampling from lakes, natural and man-made (ISO/FDIS 5667-4:2016).
Biomass and nitrogen content of plant samples from the beginning and end of the experiment were determined according to [29 ]. Briefly, plant samples were separated into roots, stems, and leaves, dried at 65 °C to a constant weight, grounded into powder, and then measured by an elemental analyzer (CHN-O-Rapid; W. C. Heraeus GmbH., Hanau, Germany) [30 (link)]. Root vitality was quantified with the triphenyl tetrazolium chloride (TTC) method [29 ]. The rate of root ROL was measured through the titanium (III) citrate buffer method [31 (link),32 (link)].

The water temperature in the mesocosms was recorded by the Temperature and Illuminance Data Logger (HOBO Pendant UA-002-08, Onset, USA). The pH was determined by a portable Multi-parameter Water Quality Meter (U-52, HORIBA, Japan). Dissolved oxygen (DO) was monitored in situ using DO electrodes (HQ40d-53 LED, HACH, USA). The concentrations of NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, TN and COD were determined with a Water Quality Analyzing System (DRB200 and DR2800, HACH, USA). All variables were analysed according to standard analytical procedures66 .

Unconditioned FS was analysed for electric conductivity (EC), pH, salinity, temperature, total solids (TS), total volatile solids (TVS), TSS, chemical oxygen demand (COD), ammonium nitrogen (NH₄-N) and nitrate nitrogen (NO₃-N). Supernatant from Imhoff cones and settling columns and leachate from dewatering columns were analysed for EC, pH, salinity, temperature, TS, TSS and COD. The analysis of solids parameters was based on Standard Methods.[29 ] TS were measured gravimetrically by drying in an oven at 105°C, and TVS at 550°C. Cellulose nitrated or glass fiber filters with a diameter of 47 mm and a pore size between 0.7 and 1.2 µm were used for TSS analysis. COD was determined with Hach vials, a Hach DRB200 heating block and a Hach DR4000v and a Dr. Lange Lasa50 spectrophotometer based on the manufacturer’s directions. EC, temperature and salinity were determined with a WTW MultiLine P4 and pH with a HANNA HI 9124 according to the manufacturer’s directions.