*3.3. Plants*

The selection of plant species has a grea<sup>t</sup> influence on the pollutant removal process. The selection of plants depends upon their local availability, the nature of pollutants, and the climate zone. The plants mostly used to develop FTWs are of *Canna*, *Typha*, *Phragmites*, and *Cyperus* genera. They have been widely applied in FTWs for the remediation of di fferent types of wastewater [30,56,61–66]. Some species of the *Poaceae* family (*Lollium* sp., *Zizania* sp., and *Chrysopogon* sp.) have been successfully applied in Italy, China, Singapore, and Thailand to develop FTWs. Some plant species are suitable for particular regions and have e fficiently removed nutrients and other pollutants in a specific climate. Some other plants such as *Phragmites*, *Carex, Acorus,* and *Juncus* were also successfully applied in FTWs, and these e ffectively adapted in several locations. The selection of macrophytes to develop FTWs is very important for pollutant removal as well as for ecosystem sustainability. The selected plants should be native, easily available, non-invasive species, perennial, able to thrive in a hydroponic environment with an extensive root system and aerenchyma [67]. The application of invasive species in FTWs may result in damage to the ecosystem, and the ultimate cost of habitat restoration may suppress the benefits gained by pollutant removal. [68]. The characteristics that make these macrophytes ideal for FTWs are their robust growth tall shoot length, extensive root system, and large aerenchyma in their roots and rhizomes. Plants with relatively thin fibrous roots have a better performance in total nitrogen removal, and plants with high total root biomass have a better performance in NH<sup>+</sup>-N removal [69]. The root development depends upon various factors such as species, age, type of plant and concentration of nutrients, trophic status of water, nature of pollutants, redox conditions, and use of supporting mats and growth media. A high nutrient load at an earlier plant stage can be harmful to plants and can damage the root system [70].

Similarly, the high load of toxicants can also hinder the growth of the root by permanently damaging young plants. The root development of *P. australis* was constrained up to 40-cm deep after 3 years of plantation due to the toxic e ffects of digestate liquid fraction. On the other hand, *Typha latifolia* and *Juncus maritimus* did not establish themselves due to the high pollutant load [71].

#### *3.4. Bacterial Biofilm*

Bacteria have a unique ability to form biofilms, also known as epiphytic microbes. Biofilm formation begins with the attachment of free-floating microbes to gas–liquid and solid–liquid interfaces. These biofilms have a key role in the assimilation of the biogeochemical cycles and the dynamics of an ecosystem process [72]. In the aquatic ecosystem, aquatic plants are an essential substrate for the establishment, growth, and development of biofilms. Aquatic plants release oxygen, essential for aerobic bacteria attached to roots, and stimulate the nitrogen cycle in the roots' surroundings [73,74]. Biofilms are composed of an extracellular matrix comprised of polysaccharide biopolymers, proteins, and DNA that hold the cell together [75]. The structural integrity of biofilms is obtained by secreted proteins, various types of exopolysaccharides and cell surface adhesions [76]. The development and maintenance of these biofilms rely on small molecules such as homoserine lactones, antibiotics, and secondary metabolites, such as the *Staphylococcus aureus* matrix, provide proteins for the synthesis of biofilm. The extracellular matrix also facilitates the formation of adhesive protein found anchored to the cell wall of *S. aureus*, holding the cells together within the biofilm by interaction with other proteins [77,78]. The extracellular DNA also strengthens the structural integrity of the biofilms. For example, *Pseudomonas aeruginosa* contains a significant amount of DNA to provide stability to biofilms [79]. The nature of biofilms and associated matrices depends upon the types of substrates, medium, and growth conditions. *Bacillus subtilis*, a Gram-positive bacterium, can make biofilms via production of two different polymers: polysaccharide extracellular polymeric substances and poly-d-glutamate. Both of these polymers contribute to biofilm formation; however, the contribution of each polymer is determined by strain and prevailing conditions [80]. The plants can also modify the function and structure of the microbial community in their rhizosphere [81]. The biodiversity and species of bacteria determine the functions of the biofilms. The biofilm-forming bacteria have been reported as diverse and host specific. The secretion of macrophytes and growth status can determine the bacterial composition of biofilms in the aquatic ecosystem [82]. Moreover, the bacterial community of biofilms was found to be different than those in the surrounding water column [37].
