CWs might be defined as engineered systems copied from natural wetlands that have been designed and constructed to utilize the natural activities involving wetland vegetation, substrate, and their associated microbial accumulation to treat nutrient-rich wastewater (Vymazal, 2007). These are artificial wastewater treatment units with shallow ponds or channels having organized plantation, which utilizes the natural biological, physical, and chemical processes to treat wastewater simulating natural wetlands within a more controlled environment (Dipu et al., 2011; Valipour and Ahn, 2016). CWs systems are fabricated and designed to get better water quality with relatively low external energy requirements, and easy operation and maintenance. This natural means of treating wastewater also offers the potential of multiple benefits, such as a source of a recreational system providing aesthetic qualities, wildlife habitats, and the superior quality effluents that can be recycled for land-scape irrigations (Iamchaturapatr et al., 2007).
The primary classification is based on the growth type of macrophytes (free-floating, floating leaves, emergent plants, submerged plants), and further classification is usually based on the water flow regime which is also depicted in Fig. 2 (Ingrao et al., 2020). According to the flow of wastewater, the CW is classified into two types, i.e., FWSCW and SSFCW (Fig. 2). The SSFCW is further classified into HSSFCW and VSSFCW. These are the most prevalently used system configurations, highlighted in (Fig. 2). The hybrid CW is the two-stage or multistage combination of HSSFCW and VSSFCW (Vymazal, 2013). The hybrid CW shows a higher nitrogen removal efficiency than the single standalone systems because it accommodates better nitrification and denitrification processes (Saeed et al., 2012; Serrano et al., 2011; Vymazal, 2005).
4.1. Parameters affecting nutrient removal efficiency of CW
The effectiveness and performance of CW technology depend on some associated factors, such as macrophytes species, microbial activity, substrate, type of wetland, DO, temperature, pH, and hydraulic loading rate, etc. (Fig. 3). The magnitude and condition of these factors at various treatment stages influence the output efficiency of technology. The factors could be divided into three categories, biotic, abiotic, and environmental factors. Biotic factors are plants and microorganisms, while abiotic factors are substrate, precipitation, evaporation, etc., used in CW. DO, temperature, and pH are the main environmental components in a CW (Jingyu et al., 2020; Sindilariu et al., 2009). Many biological processes of CW are affected by DO and temperature (Oliver et al., 2017).
4.1.1. Dissolved oxygen and artificial aeration
Nitrification or denitrification processes can be controlled by the oxygen level in the CW (Nuamah et al., 2020; Wu et al., 2014). The presence of oxygen is affected by plant respiration and microbial activities (Shelef et al., 2013). NH4+-N to NO3−-N conversion rate increase significantly by continuous oxygen supply or aeration. Sometimes an electromechanical device is used for a continuous supply of air (Foladori et al., 2013). Fan et al., (2013) found that TN and NH4+-N removal efficiency was 63% and 58%, respectively without aeration, and 82% and 96% respectively, with artificial aeration and step feeding strategy in VSSFCW due to the formation of alternative aerobic and anaerobic zones and increase in the heterotrophic bacterial activity. Different types of aeration systems were used in the CW, such as step feeding, continuous aeration, intermittent aeration (Fan et al., 2013). It was reported that CW operating with intermittent aeration followed by step feeding resulted in removal efficiencies of NH4+-N and TN to be 96% and 82%, respectively, at 14.7°C (Ji et al., 2020). Recently, the use of artificial aeration is becoming popular in CW technology (Jizheng et al., 2019). In the HSSFCW, oxygen depletion is more common, and denitrification is the dominating process (Xinshan et al., 2010). Artificially aerated CW achieved significantly higher NH4+-N, and TN removal efficiency, with elevated microbial abundance than non-aerated ones (Yang et al., 2018). However, the removal efficiency of TP was not very much affected by the artificial aeration. Rossmann et al., (2012) reported that TN removal is enhanced by 12% while, TP increased only by 6% with artificial aeration. It was also reported that for 0.6 L/min aeration rate for 2 h/day, the removal rate of TN, NH4+ N, and COD were 78%, 97%, and 96%, respectively (Zhou et al., 2018).
The treatment performance of CW is affected by temperature variations. It affects both the biological reactions and physical processes in the wetland systems. Many biological processes regulate the removal of nutrients, which are controlled by temperature, affecting overall efficiency (El-Refaie, 2010). Evaporation water loss affects nutrient removal because the chemical budget is associated with the water budget in a CW (Lott and Hunt, 2001). El-Refaie (2010) found a direct relationship between temperature and phosphorus removal. It was reported that TP removal efficiency in winter was 10%, while in summer, it was 66.7%. A similar trend was also obtained for nitrogen removal (Mesquita et al., 2017). Nitrogen removal is more influenced by temperature variations than phosphorus; as most phosphorus removal mechanisms depend upon the physicochemical and sorption processes. In contrast, nitrogen removal depends on microbial activities as the microbes are temperature sensitive (Picard et al., 2005; Spieles and Mitsch, 1999). Temperature also affects oxygen availability or indirectly decreases the redox process (Bachand and Horne, 1999; Kadlec, 1999). Redox processes are responsible processes for phosphorus adsorption on ferrous and ferric oxide, which results in a decrease in the removal efficiency of phosphorus with decreasing temperature (Wittgren and Maehlum, 1997). This study observed that in the summer season (temperature range 23oC − 29oC) the removal efficiency of NH4+-N, TN, NO3-N, and TP increased by 25.4%, 28.16%, 28.5% and 28.35% respectively when compared to winter season summer season (temperature range − 11.1oC to 14oC) (Supplementary Table A.2).
4.1.3. pH Level
The performance of CW is affected by pH as it directly or indirectly influences the biotic and abiotic factors of the CW. The organic nitrogen conversion processes i.e. ammonification, nitrification and denitrification are pH-dependent reactions (Parde et al., 2021; Vymazal, 2007). Intracellular metabolic activity, cell growth, and biomass may be affected by pH (Çelen et al., 2007; Ranieri et al., 2013). Even the increased pH level can reduce the oxygen concentration in sediments (Yin et al., 2016). It was reported that the TN removal was decreased from 76.3 to 51.8% at stress pH level (from 7.5 to 10.5) and the NH4+-N and NO3−-N removal were also adversely affected. The stress in pH was due to plant assimilation and decay in the summer season (Yin et al., 2016). In summer, the pH level of submerged planted CW increased rapidly, creating stress for emergent plants (Mjelde et al., 2012). The pH level is high in the summer season because of the intensive photosynthesis activity of submerged plants and high oxygen transfer from macrophyte to CW (Yin et al., 2016). It was reported that the heterotrophic bacterial activity was the highest at neutral pH and reduces with pH variations (Meng et al., 2014). The optimal pH range for nitrifying bacteria was 7 to 8 (Antoniou et al., 1990; Painter and Loveless, 1983; Paredes et al., 2007). The pH range of 6 to 8 is ideal denitrification activity; however, the reaction rate also decreases when the pH reduces below 5, and even it becomes negligible below pH 4 (Vymazal, 2007). It was reported that at a higher pH level of approximately 10 to 11 plant growth rate started reducing (Hadad et al., 2018). The protoplasm of many vascular plants' root cells was seriously damaged at pH above 9 and in low pH (4.3 to 6.2), the free-floating species were about to die (Akçin et al., 1993; Dyhr-Jensen and Brix, 1996). T. latifolia showed a relatively low growth rate at low pH of 3.5 as compared to 5, because at pH of 3.5, the H+ ions concentration increased, and the electrochemical gradient decreased across the plasma membranes of the root cell (Dyhr-Jensen and Brix, 1996). It was reported that the soil, plant, microorganism, and phosphorus release was also influenced by changes in pH (Kim et al., 2016).
Soil, rock, gravel, and organic materials are common substrates used in CW (Ballantine and Tanner, 2010). These substrates can be divided into three types concerning their origin, viz. naturally occurring materials (soil, sand, gravel, etc.), processed materials (alum, amended zeolite, ceramics etc.), and waste materials (fly ashes, slag, etc.) (Ballantine and Tanner, 2010; Dong et al., 2021). Nitrogen removal in CW can be obtained satisfactorily with the help of the microorganism and substrates, but phosphorus removal is obtained mainly through the adsorption process on substrates (Cui et al., 2008). Nguyen et al. (2020) depicted major phosphorus removal by adsorption (77.5%), followed by microbial assimilation (14.5%), plant uptake (5.4%), and other processes (2.6%). (Xu et al., 2006) Xu et al. (2006) reported that the sorption capacity varies with the types of substrate materials. Fine sized substrates have more surface area, which attributes to more phosphorus adsorption capacity; thus, the phosphorus removal efficiency can be increased by choosing a better substrates material and particle size (Cui et al., 2008; Xu et al., 2006). (Lima et al., 2018) Lima et al. (2018), stated that on treating low-strength sewage with E. crassipes, the phosphorus removal efficiency in a CW with gravel substrate was 25%, while with bricks, it was 87%. In another study by Vymazal (2004), it was reported that in the long term, more than 95% of phosphorus is stored in soil or litter only. Other than phosphorus adsorption, the substrates also support living organisms, microbial transportation and regulates the water flow through the CW.
Bacterial diversity living inside plant tissues is responsible for several physicochemical and biological processes occurring in the system (Rajan et al., 2019). Thus, microbial activity is the critical factor influencing the proper functioning and maintenance of the CW (Ibekwe et al., 2003). It was reported that microbes play an essential role in organic and inorganic pollutant removal and plant growth (Shahid et al., 2020). Microorganisms are responsible for the transformation and mineralization of nutrients (Dinakar et al., 2020; Stottmeister et al., 2003). Various types of processes, such as ammonification, nitrification, denitrification, and nitrogen fixation are depended upon the activity and diversity of the microorganisms (Bañeras et al., 2012; Bell et al., 2005). It was reported that the presence of various types of bacteria, such as Nitrosopumilus, Vibrio, Pseudoalteromonas, Nitrospina, and Planctomyces contribute to effective nutrient removal and plant growth in CW (Ma et al., 2018). Various bacteria are responsible for specific functions, such as Syntrophusand, Syntrophobacter bacteria responsible for nitrification, whereas Pseudomonas, Dechloromonas, Desulfomicrobium, and Desulfobacca are denitrifying bacteria (Li et al., 2020). Lee et al. (2009) reported that a major portion of the nitrogen is removed by the denitrification process, which decreased about 60 to 70% of the nitrogen while around 20 to 30% was removed by plant uptake.
Phosphorus is degraded by microbes in the form of orthophosphate along with many other organophosphorus compounds, while its uptake is limited due to less storage capacity (Singh and Walker, 2006; Vymazal, 2007). Microorganisms attain nutrients from the root exudates of macrophytes which is one of the key factors affecting the removal efficiency of nutrients in CW.
4.2. Assessment of the role of macrophytes in CW
Macrophytes or plants are responsible for providing the necessary components for nutrient removal, such as the development of microbial communities near plant roots and fulfilling the oxygen requirement of wetland (Rossmann et al., 2012). Macrophytes play a vital role in CW processes, especially for nutrient removal via direct plant uptake and provide the substrate for microbial activities (Meng et al., 2014). All microbial activities, density, and diversity are supported by the plant rhizosphere (Carvalho et al., 2014; Truu et al., 2009). The enzyme activities play an important role in microbial production, which are also affected by root and shoot. Planted CWs have more soil microbes and redox activities than unplanted wetlands (Huang et al., 2012; Salvato et al., 2012; Tanner, 2001). Some principal features, such as root morphology, structure, and eco-physiology, are influenced by plants, and these features have an important role in nutrient retention and enzyme activities (Zhang et al., 2010). Even dead plants can produce useful organic compounds, i.e., amino acids, sugars, and volatile fatty acids, that will help in the growth of plants and microbes which increases contaminants removal efficiency (Vymazal, 2007).
It was reported that with the vegetation growth and adequate carbon availability, the denitrification process increased, and phosphorus removal processes also increased (Craft, 1996; Kadlec, 1999). Wetland macrophytes supply oxygen to the root zone which influences the aerobic activities in CW and reduce up to 90% of BOD (Bodelier et al., 1996; Moorhead and Reddy, 1988). Individual plant species have different capacities of oxygen supply to the root zone according to their ability, vascular tissues, root distribution, and associated metabolic activities (Shimamura et al., 2003). The root zone is also known as the rhizosphere, and it is divided into two parts endorhizosphere (the root interior), and the ectorhizosphere (the root exterior). The interaction area of these two zones is known as the rhizoplane, and this is the main place of interaction between the plant and microorganisms (Stottmeister et al., 2003).
Figure 4 summarizes salient features and various roles of macrophytes in CW with nitrogen and phosphorus removal mechanisms. The major role of plants in CW are related to its hydraulic function (control flow), physical functions (fixing substrate, providing shelter to microbes), environmental functions (maintain DO level), and most important treatment of nutrients by plant uptake, degradation, transport, transform, store, and phytovolatilization of nutrients. Sometimes plants also contribute to nutrient input in CW through litter fall, which may also affect the total nutrient input in the CW system (Shelef et al., 2013).
A critical function of plant roots is to maintain sediments' hydraulic conductivity (Brix, 1994; Wittgren and Maehlum, 1997). Other than treatment facilities, it also provides green space and provides shelter to many aquatic animals with a good aesthetic appearance. A study described that temperature and oxygen release in root zones are affected by plant species which alters overall wastewater treatment efficiency (Allen et al., 2002). Vascular plants or non-vascular plants (algae) are an essential part of a CW. Algae enhance the dissolved oxygen of water which indirectly increases the nutrient removal reactions. Plants properly control the water flow and spread it throughout the substrates of the CW. Moreover, they uptake nutrients, carbon, and other contaminants and fix them in their tissues. The roots of the macrophytes provide a surface for microorganism activities. Several factors, such as types of CW, quality, and quantity of wastewater, affect system’s removal efficiencies (Sklarz et al., 2009). Zhou et al. (2017) assessed the effect of vegetation on the treatment of domestic sewage in HSSFCW and found that the NO3−-N removal rate is 99.9% in a vegetative CW, much higher than an unplanted wetland (82.9%).
In a substrate-free CW, Kyambadde et al. (2005) obtained the TN and PO43−-P removal efficiency in planted CW to be 73.9% and 73.5%, while in an unplanted CW, they were found to be 54.3% and 45.2%, respectively. Evapotranspiration of emergent macrophytes is an essential activity in a CW (Shelef et al., 2013). It was reported that the water losses by evapotranspiration reduce the flow velocities, increase retention times and enhance pollutant concentration in water (Headley et al., 2012). Microclimatic conditions may affect different components of CWs, i.e., the shades of plants can obstruct algal growth, protect from direct radiation, and slow down wind velocity that helps in stabilizing the upper substrate (Shelef et al., 2013). Although plant growth is high in the summer season due to the high level of solar radiation and high relative humidity. In autumn, plant leaves dry and therefore, the overall nutrient removal rate is higher in summer and lower in autumn (Licata et al., 2019). The biomass produced by the plants have various commercial uses and its production is mainly obtained by pruning and harvesting operations of plants and used as fodder for livestock, fertilizer, and sometimes as biofuel (Licata et al., 2019; Liu et al., 2012). The planted wetland may be an effective carbon sink and help in environmental production (Mander et al., 2008; Mitsch et al., 2013). Plants enhance nitrogen removal by providing stem and root surface area for microorganisms and lowering the algal growth (Oliver et al., 2017).
4.2.1. Difference between monoculture and polyculture practices in CW
Effluent quality is affected by the presence of different plant species in the wetland (Iamchaturapatr et al., 2007; Maine et al., 2007). Every plant has a specific capacity that influences nitrogen removal in CW, which is also affected by species richness, i.e. number and type of species (Zhu et al., 2012). It was reported that suitable macrophyte selection could have an important role in improving removal efficiency for different types of contaminants (Brisson and Chazarenc, 2008). Monoculture and polyculture (mixed culture) are the two types of planting practices in CW. Monoculture is the practice of growing a single type of plant species (such as Typha latifolia plantation), while polyculture refers to planting multiple types of plant species (such as Typha latifolia plantation with augustifolia or other plant species) in CW (Fig. 5. a & b).
In most of the CW studies, monoculture is practised, but it was reported that the plant diversity could increase the plant’s effectiveness for better contaminant removal (Liang et al., 2011). Zheng et al. (2016), reported that NO3−-N, NH4+-N, and TP removal efficiency is higher in vegetated CW than unplanted ones. Plant diversity also helps in better system functioning because competition between plant species results in more biomass production, less dominance of a single species, and reduced sensitivity to seasonal changes (Qiu et al., 2011). Better root growth and more microbial diversity were observed in the polyculture (Amon et al., 2007). Due to more release of root exudates in polyculture, the nutrient removal via plant uptake might be enhanced (Marín-Muñiz et al., 2020; Wu et al., 2012). Polyculture has shown higher nutrient removal efficiencies (Supplementary Table A.1), this might be a result of the fact that different macrophyte species have specific nutrient selectivity for plant uptake, growth rates, and microbial processes (Liang et al., 2011; Zhang et al., 2009). However, due to the similar characteristics with comparable growth and feed demand, the competition between different plants in polyculture may be more (Engelhardt and Ritchie, 2001; Zhang et al., 2007b). Liang et al. (2011) found that the plant density in polyculture (123.8 individuals/m2) was higher than in monoculture (71.3 individuals/m2). The same study reported that in the first year of planting, the biomass production in monoculture (2041.8 g/m2) was more than in polyculture (1703.7 g/m2). Moreover, after three years, the result was changed, and biomass production in polyculture (1661.5 g/m2) was higher than in monoculture (1306 g/m2) (Liang et al., 2011). The growth of polyculture is higher because it reduces the negative effect of some species and becomes more resilient to diseases (Liang et al., 2011). Geng et al. (2019) reported that the nutrient selection and the nutrient removal process, i.e., nitrification, denitrification, and plant uptake, also changed with plant species. The study also reported that all types of nitrogen removal efficiencies were better in polyculture than monoculture (Geng et al., 2019).
Figure 5c & d are showing the mean nutrient removal efficiencies for NH4+-N, TN, NO− 3-N, PO43−-P, TP in CW in monoculture and polyculture practices. Average nutrients removal rates in HSSFCW for TN, NO3−-N, TP were found to be 88.55%, 84.06%, 74.34%, respectively in monoculture (Supplementary Table A.1). While for VSSFCW in monoculture CW system, nutrients removal rates for TN, NO3−-N, TP were 60.11%, 87.21%, 58%, respectively (Supplementary Table A.1). In the case of polyculture, the average removal efficiencies of HSSFCW were found to be TN, NO3−-N, TP were 95.26%, 89.5%, 79.17% respectively (Supplementary Table A.1). While VSSFCW achieved a removal efficacy of around 81%, 71.6%, and 92.8% for TN, NO3−-N, TP respectively (Supplementary Table A.1).