**2. Methodology**

#### *2.1. Diesel Oil*

The diesel oil used in this study was purchased from a local filling station. The diesel oil was filter sterilized via syringe filters and was used as such without any further analysis for detail composition of the entire compounds.

#### *2.2. Preparation of Mixed Bacterial Culture*

Three hydrocarbons degrading bacterial strains viz.; Acinetobacter sp.61KJ620863, Bacillus megaterium 65 KF478214, and Acinetobacter sp. 82 KF478231 already isolated and characterized [24] were applied in the current experiment. All the hydrocarbons degrading bacterial strains were grown separately in M9 solution containing diesel oil 1% (w/v) as a sole carbon source. After growth in M9 solution, the bacterial culture was centrifuged then resuspended in normal saline solution. To achieve 10<sup>8</sup> cells mL−1, the optical density of each microbial suspension was adjusted finally using normal saline solution. The bacterial suspension was spread on the LB agar plates to count the number of colonies. The cell suspension of each bacterial strain was mixed in equal proportion and the consortium (50 mL) was inoculated in FTWs microcosms.

#### *2.3. Manufacturing of Floating Treatment Wetlands*

The FTWs were fabricated at the National Institute for Biotechnology and Genetic Engineering, (NIBGE) Faisalabad Pakistan. The experiment was conducted for 90 days beginning in the month of April 2018. In the FTWs microcosms, the tanks having 20 L volume capacity were made of poly ethylene material, whereas the floating mats were preapared using Jumbolon sheets of Diamond Foam Company, Pvt. Ltd., Pakistan. The Jumbolon foam piece, which was used as a floating mat, comprises the dimension of 50.8 cm (length) × 38.1 cm (width) × 7.62 cm (thickness) and five holes in each mat which were drilled at equal distance for growing healthy seedlings of *Cyperus laevigatus* L.

The seedlings of selected plant were allowed to grow for thirty days duration in the FTWs microcosms containing tap water without any treatment. However, Hoagland solution was applied periodically to stabilize the process of root-establishment. After thirty days acclimatization of plant roots, the outer surface of roots was sterilized using 5% NaOCl (sodium hypochlorite) solution. The established microcosms were spiked with 1% diesel oil (w/v). The concentration of diesel oil was selected based on our previous studies of diesel oil biodegradation (0.5%, 1.0%, and 1.5% diesel oil) in M9 media by shake flask experimentation.

## *2.4. Experimental Design*

The experimental setup of floating treatment wetlands is shown in Figure 1. The floating treatment wetland microcosms were prepared in triplicates and di fferent types of treatments were as follows:

**Control-1 (C1)**: Microcosm consist of diesel oil polluted water and no plants.

**Control-2 (C2)**: Microcosm consist of tap water and plants.

**Treatment-1 (T1)**: Microcosm consist of diesel oil polluted water and bacterial consortium.

**Treatment-2 (T2)**: Microcosm consist of diesel oil polluted water and plants.

**Treatment-3 (T3)**: Microcosm consist of diesel oil polluted water, plants, and bacterial consortium.

**Figure 1.** Experimental setup showing the floating treatment wetlands system for the cleanup of diesel oil polluted water.

## *2.5. Plant Biomass*

After 3 months of the experimental period, the plant roots and shoots were cropped 2 cm down and overhead the floating mat. For both roots and shoots, the fresh and dry biomass was determined to check the influence of microbial inoculation and plant growth on diesel oil remediation. Fresh roots and shoots samples were placed in an oven at 70 ◦C and dry biomass was determined [25].
