## 2 Materials and Methods  
### Microbial Isolation  
Twenty (\(n=20\)) morphologically different bacterial microorganisms were isolated from swabs and the poultry slaughterhouse wastewater (PSW) collected from the drainage port of a poultry slaughterhouse in Cape Town, Western Cape, South Africa, which slaughterts about 1 million birds per week, and then stored at 4 \({}^{\circ}\)C before use. The samples were subjected to repeated dilution, resulting in a dilution factor of \(\times 10^{8}\). This approach was used to enhance the prospect of obtaining distinct colonies. A volume of 100 \(\mu\)L obtainedfrom the preceding four dilutions was used for spread plating onto nutrient agar (31 g/L, Biolab, Diagnostics Laboratory Inc., Budapest, Hungary). The plates were inverted and incubated at 36 \({}^{\circ}\)C for 24 h. After the incubation period, colonies were distinguished based on their physical appearance, which included color, appearance, texture, and form. Different and single colonies were picked and subcultured onto fresh nutrient agar plates using a streak plating technique. Continued sub-culturing occurred until pure cultures were obtained [15]. The pure culture plates were coded and numbered in alphabetical order. Bacterial isolates were screened for biofloculant production using a standardized method by determining flocculation activity [16]. The isolate with high flocculation activity was characterized using morphological features (structure, color, Gram reaction) with an Olympus light microscope (Olympus CX21 FS1, Tudortech Pty Ltd., Bryanston, South Africa) at a \(\times\)100 magnification. Isolate D2 was the selected strain and was used for further experiments.  
DNA was isolated from the bacteria (isolate D2) using Zymo's Bacterial DNA extraction kit (Zymo Research LLC, California, United States of America) and a Thermo Fisher Scientific (Johannesburg, South Africa) DreamTaq(tm) DNA polymerase and universal primers 27F (5\({}^{\prime}\) AGAGTTTGATCMTGGCTCAG 3\({}^{\prime}\)) and 1492R (5\({}^{\prime}\) CGGTACCTTGTAC-GACT 3\({}^{\prime}\)) for forward and reverse reactions, respectively, were then used for amplification of the target 16S region. The amplicons were gel extracted using Zymo's Zymoclean(tm) Gel DNA recovery kit (Zymo Research LLC, Tustin, CA, USA) and sequenced by the ABI PRISM(tm) 3500 xl Genetic Analyzer (Thermo Fisher Scientific, Johannesburg, South Africa). The DNA was cleaned to remove post-cycle sequencing reaction contaminants using Zymo's ZR-96 DNA Sequencing clean-up kit (Zymo Research LLC, Tustin, CA, USA), followed by sequence analysis using a QIAGEN CLC Main Workbench v.7 (QIAGEN Sciences LCL, Germantown, MD, United States of America). The resultant 16S rDNA was used to determine the most similar sequences using the Basic Local Alignment Search Tool (BLAST) via the National Centre for Biotechnology Information (NCBI) databases ([[https://www.ncbi.nlm.nih.gov/](https://www.ncbi.nlm.nih.gov/)]([https://www.ncbi.nlm.nih.gov/](https://www.ncbi.nlm.nih.gov/))) [Accessed on 22 April 2022]. For RpoD, the primer sequence for the targeted region was as follows: RpoD-F 5\({}^{\prime}\)ATGCCAAAACGGTATGTCG3\({}^{\prime}\) and RpoD-R 5\({}^{\prime}\)TCTTGTACGRCCRTCATCAAG3\({}^{\prime}\) (Zymo's Bacterial DNA extraction kit, Zymo Research LLC, Tustin, CA, USA), and the same procedure was followed.