*2.2. Macrophytes*

The *Phragmites australis* commonly known as common reed was used to carry out this research. It was selected because it has previously proven its effectiveness in reducing the toxicity of polluted wastewater in different studies [11,25,31]. The *P. australis* has an extensive root and shoot system that helps in better oxygen supply to the root zone, thereby enhancing the bacterial propagation and increased pollutants degradation [32].

#### *2.3. Endophytic Bacterial Strains*

In this study a consortium of three bacterial strains was applied, namely *Rhodococcus sp.* (NCBI Accession: MF326802), *Pseudomonas indoloxydans* (NCBI Accession: MF478985) and *Acinetobacter junii* (NCBI Accession: MF478980) [25,30]. The strain *P. indoloxydans* was endophyte because it was isolated from the root interior of *Polygonum aviculare*. The strain *Rhodococcus sp.* was rhizospheric as it was isolated from the rhizosphere of *Poa labillardierei*, and the strain *A. junii* was isolated from activated sludge [28]. These specific bacterial strains were chosen due to their potential to reduce textile dyes and assist the macrophytes to alleviate pollutant-induced toxicity without compromising plant growth and development.

The bacterial strains were cultured as separate cultures at 30 ◦C for 24 h in Luria–Bertani (LB) broth. The bacterial cell pellets were isolated by centrifugation at 4 ◦C, followed by resuspension in 0.9% NaCl solution [25]. The optical density of each bacterial inoculum was adjusted to 0.9 at 600 nm according to the guidelines of the turbid metric method [33]. The bacterial consortium (10<sup>8</sup> colony forming units (CFU) mL−1) was prepared by mixing all bacterial inoculum together in equal proportion. This bacterial consortium was used as an inoculum to inoculate the floating treatment wetlands.

#### *2.4. Fabrication of FTWs and Experimental Setup*

The macrocosms experimental setup was comprised of nine tanks with 1000 liter capacity each, and the dimensions were 1.2 m (L × W × H). The tanks were painted black form all sides to minimize the algal growth. The floating mats were fabricated from expanded polystyrene (EPS)-based sheets manufactured by Diamond® Foam Private Ltd., Pakistan [11,34–36]. EPS sheets are rigid, have low thermal conductivity, are moisture resistant and consist of non-porous closed cell foam [37]. The size of the floating mats was adjusted so that they could float in each tank with >95% coverage on the water surface. All four sides of the floating mats were wrapped with aluminum foil to protect the sheets from sun and water damage. In each floating sheet eight equidistant holes, equal in diameter, were made for the plantation of macrophytes on the floating mats. Each hole was planted with three healthy seedlings of *P. australis*, thus having 24 seedlings in each mat. Each seedling weighed 45 to 65 g and their length was 55–65 cm. The seedlings were supported by coconut shavings and soil in the floating mat. The seedlings were allowed to grow in fresh water for one month to gain optimum growth of roots and shoots. After one month, the average height of the plants was about 145 cm, and the fresh water in tanks was replaced with the synthetic textile effluent enriched with dyes. The experiment was run in triplicate with the subsequent treatment design:

T1D1, T1D2, T1D3: Only dye;

T2D1, T2D2, T2D3: Containing dye and plants;

T3D1, T3D2, T3D3: Dye, plants and bacterial consortium;

T4: Fresh water and plants.

(D1: Bemaplex Navy Blue DRD, D2: Bemaplex Rubine DB, D3: Bemaplex Black DRKP Bezma).

The treatments T3D1, T3D2, T3D3 were inoculated by pouring one liter of inoculum into each tank. The experiment lasted for 20 days until a maximum of dye and pollutants were removed from treated water. One liter of sample was collected from each tank every 5 days starting from day 0 using a sequencing fill-and-draw batch mode method (for convenience, data of only the 0, 10th and 20th day are presented in results). The samples were stored in a cool and dry place for further analysis [38]. The collected water samples were analyzed for pH, electrical conductivity (EC), total dissolved solids (TDS), total suspended solids (TSS), dye concentration, chemical oxygen demand (COD), biological oxygen demand (BOD), colony forming unit (CFU) and metal concentration (Cu, Fe, Mn, Ni, Zn and Pb) according to standard methods [38]. The evapo-transpiration losses were recovered by pouring fresh water in treatment tanks up to the level of 1000 L in each tank [34]. In case of rain, the tanks were covered with plastic sheets.

#### *2.5. Persistence of Inoculated Bacteria in Treated Water and Plants*

The persistence of bacteria in water, root and shoot samples were periodically analyzed during the experiment using the cultivation-dependent plate count method [24,25]. The collected roots and shoots samples were surface sterilized by 70% ethanol and 2% sodium hypochlorite solution. Then these roots and shoots were homogenized in a 0.9% NaCl solution and serial dilution of these suspensions was spread on LB agar plates. Similarly, the collected water samples from all treatments were spread on LB agar plates and these plates were incubated at 37 ◦C for 48 h for CFU analysis [35,39].

#### *2.6. Plant Biomass*

In order to determine the e ffect of bacterial inoculation and dye-induced toxicity on plants growth and development, the data about plants agronomic parameters (root and shoot length and dry biomass) were noted at the end of the experiment. The root and shoot length was measured manually by a measuring scale. The root and shoots were harvested near the surface of the floating mat and oven dried at the 80 ◦C for 72 h until a constant weight was achieved [11,34].

## *2.7. Statistical Analysis*

The results of physicochemical parameters (pH, EC, TDS, TSS, BOD, COD, color and heavy metals), bacterial persistence and plant biomass were evaluated by the SPSS software package. The comparison between treatments was executed by analysis of variance (ANOVA) followed by a Post-Hoc Tukey test (*p* ≤ 0.05) [40]. The alphabet labels over the values show the significant/non-significant di fferences among treatments.
