Abstract This study develops an innovative vertical up-flow constructed wetlands (VUF-CWs) design that improves oxygen distribution and nutrient removal using porous substrate (biochar, pumice) and intermittent water recirculation. The lab-scale systems planted with Canna indica L. under three water recirculation regimes (no recirculation, 3:3 h, and 6:3 h recirculation/non-recirculation) were compared with unplanted systems. The results of 3-day retention time operation showed that the planted systems operated under the 6:3 h recirculation/non-recirculation regime achieved the highest dissolved oxygen (DO) concentration (3.9 mg L^-1) and the highest removal of BOD₅, NH₄⁺-N, and NO₃^--N (approximately 73%, 73%, and 90%, respectively), while maintaining moderate PO₄^3--P removal (approximately 20%). Meanwhile, DO concentration was limited to 0.6 mg L^-1 without plants or recirculation, hence, low removal of BOD₅, NH₄⁺-N, and NO₃^--N (approximately 8%, 26%, and 24%, respectively). Nitrogen removal and organic degradation were significantly enhanced by the combined effects of plant oxygenation, oxygen preservation in biochar-pumice media, and improved mixing through intermittent recirculation. However, prolonged recirculation time reduced the removal of total suspended solids (TSS). The findings demonstrate a synergistic mechanism between plant oxygen release, porous substrate function, and intermittent recirculation, providing an effective strategy with simple design to improve performance of a nature-based wastewater treatment system.   

Keywords: Biochar, Nature-based solutions, Vertical up-flow, Wastewater treatment

Citation:  Thompradit, K., Pincum, T., and Jampeetong, A. 2026.  Enhancing oxygen transfer and nitrogen removal in vertical up-flow constructed wetlands using intermittent recirculation and porous media with Canna indica. Natural and Life Sciences Communications. 25(4): e2026088.

Graphical Abstract:

INTRODUCTION

Many wetlands have been impacted by ongoing population growth in recent years owing to human activity-induced pollutant contamination. Excess nutrients can promote harmful algal blooms, which typically result in water quality deterioration and decreased oxygen levels due to decomposition processes (Amorim and Moura, 2021). Constructed wetlands (CWs) are artificial systems that mimic the wetland’s natural function to reduce nutrients and other pollutants through many processes (Vymazal, 2022). In CWs, suspended contaminants are removed through physical processes such as filtration, whereas nutrients and other organic matters are removed through biological processes including plant uptake, microbial biodegradation, and nitrification-denitrification (Vymazal, 2010; Rout et al., 2021). As nature-based treatment systems, CWs can effectively improve water quality while requiring relatively low energy and operational inputs.

The use of CWs depends on several criteria, for example, types of wastewater, area availability, system design, and their efficiency (Shukla et al., 2022). Regarding treatment performance, vertical flow constructed wetlands (VFCWs) generally provide better oxygen transfer than horizontal flow constructed wetlands (HFCWs), which promote aerobic biodegradation and nitrificaion (Vymazal, 2022; Santos et al, 2024). Vymazal (2010) reported that NH₄⁺-N removal efficiency was higher in VFCWs (73%) compared with HFCWs (30-39%), indicating a greater potential for nitrification due to their high oxygenation capacity. Similar findings have also been reported in other studies (Soundaranayaki and Gandhimathi, 2019; Thalla et al., 2019). Nevertheless, vertical downflow systems are prone to clogging when the systems receive a high load of suspended solids and particulate matter, as solids accumulation can eventually reduce treatment efficiency (Pucher and Langergraber, 2019). Furthermore, deeper layers frequently have lower oxygen levels than the surface and root zones, which can restrict aerobic degradation processes (Shukla et al., 2022). Vertical up-flow constructed wetlands (VUF-CWs) have been suggested to increase wastewater retention time, thereby enhancing interactions among wastewater, substrates, biofilms, and the rhizosphere, which may improve nutrient removal (Cui et al., 2010; John et al., 2020). This configuration may also help mitigate clogging risk by promoting a more even distribution of solids within the substrate. However, nitrification in VUF-CWs may be limited (Zhao et al., 2011), which could be related to low oxygen availability.

In biological wastewater treatment systems, oxygen plays a crucial role facilitating nutrients and organic matters removal (Vymazal, 2010). Current research focused on improving oxygen transfer has been growing interest through innovative design such as flow-direction control, water circulation and substrate selection (Zhao et al., 2011; Foladori et al., 2013; Chand et al., 2022; Xiao et al., 2023). Vertical flow CWs combined with intermittent water recirculation have shown enhanced oxygenation and improved treatment performance compared with conventional systems (Foladori et al., 2013). Baggiotto et al. (2025) found that vertical flow constructed wetland operated with recirculation had higher NH₄⁺-N removal than the system operated without recirculation due to enhanced nitrification resulting from increased oxygen transfer. Therefore, water recirculation may be an effective strategy to overcome low oxygen availability in VUF-CWs and promote coupled nitrification and denitrification. In addition, porous substrates such as biochar and pumice have specific properties that can enhance pollutant removal. Biochar derived from agricultural residues also promotes resource recovery from biomass waste. In northern Thailand, longan cultivation generates substantial quantities of residues that can be converted into value-added biochar products. A previous study found that longan biochar has a high specific surface area (SSA) and pore volume (PV), which are effective for NH₄⁺-N removal and oxygen preservation, respectively (Janyasupab and Jampeetong, 2022). Therefore, integrating a vertical upflow system with intermittent recirculation and porous substrates such as biochar is expected to improve oxygen availability and transfer and to enhance treatment performance, particularly for nitrogen removal.

Canna indica L. is a common ornamental wetland plant widely distributed in Southeast Asia. This species can grow and adapt well even in polluted water due to its high root biomass and large root surface area, which effective uptake of nutrients and other pollutants. Several studies have demonstrated the potential of C. indica to remove BOD₅ and nutrients in various constructed wetland systems (Pinninti et al., 2022; Phewnil et al., 2024; Sharma et al., 2024). Moreover, C. indica develops aerenchyma in its root cortex, facilitating oxygen transport from shoots to roots and releasing into the rhizosphere, thereby creating an aerobic environment for the nitrification process (Suriyakaew and Jampeetong, 2021). Currently, porous substrates are applied in CWs because of their potential for oxygen preservation. A study by Jampeetong and Janyasupab (2022) found that biochar and pumice enhanced root diameter and elongation, resulting in higher nitrogen removal efficiency. However, negative effects of biochar on plant growth have been reported (Janyasupab et al., 2023). To minimize such negative effects of biochar, this study designed a system by growing C. indica on gravel as a basal substrate, followed by a layer of pumice and biochar. The up-flow design was applied to allow the filtration of suspended solids and organic matter by porous substrates prior to nutrients uptake by plant. Intermittent water recirculation was also applied to enhance oxygenation and overall removal efficiency.

Despite increasing interest in improving oxygenation and nutrient removal in constructed wetlands, research integrating intermittent recirculation with porous substrates in VUF-CWs remain limited. To address these gaps, we developed an innovative intermittent circulating VUF-CW system that combines biochar-pumice media with intermittent up-flow recirculation and C. indica planting to maximize oxygen distribution and pollutant removal. Beyond improving treatment performance, the proposed system supports United Nations Sustainable Development Goal 6
(SDG 6; Clean Water and Sanitation) through enhanced wastewater treatment performance while promoting circular economy through the beneficial reuse of agricultural waste-derived biochar as a treatment medium.

MATERIALS AND METHODS

Experimental setup

Laboratory-scale VUF-CWs (19.5 cm in width, 19.5 cm in length, and 60 cm in height) were created from transparent acrylic sheets (3 mm thick). Each unit was placed over aluminum foil to prevent light penetration. Longan biochar (Ø 3–5 mm), pumice (Ø 7–12 mm), and gravel (Ø 10–15 mm) were arranged in layers from bottom to top, with the depth of 15 cm for each layer (Figure 1). The biochar derived from longan branches was obtained from Warm Heart Foundation (Phrao district, Chiang Mai Province, Thailand) and sieved through a 1.5 mm mesh to remove ash particles before use. Detailed physicochemical properties, including specific surface area, pore volume, and cation exchange capacity (CEC) were reported previously by Janyasupab and Jampeetong (2022). The simulated VUF-CWs were either unplanted or planted with C. indica. Approximately one-month-old plants of similar size were selected and planted into the upper substrate layer of each unit. A DC mini water pump (Input 6–12 V) was installed, and the water flow rate was set to 2.5 L min^-1.

Figure 1. Schematic diagram of recirculating VUF-CW systems planted with C. indica and unplanted systems, operated under 0:24 h (no recirculation), 3:3 h, and 6:3 h (recirculation/non-recirculation) regimes.

Each system was filled with 8 liters of real wastewater collected from the effluent of a biogas system treating wastewater from poultry and swine farms at Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University (Mae Hia Campus), Chiang Mai, Thailand. Before being used in the experiment, the biogas effluent was diluted four-fold with tap water to achieve an appropriate influent strength and prevent excessive loading to the treatment units. The characteristics of the diluted wastewater used in this study are summarized in Table 1. Each system was acclimatized for 2 months to allow plant growth and biofilm formation. Both the unplanted and planted systems were refilled with wastewater. For each treatment, four replicates were operated under up-flow conditions with intermittent water recirculation regimes: 0:24 h (no recirculation), 3:3 h, and 6:3 h (recirculation/non-recirculation). The schematic of the vertical up-flow constructed wetland (VUF-CW) system is shown in Figure 1. This study was conducted in the greenhouse of the Department of Biology, Faculty of Science, Chiang Mai University, Thailand. The average day/night temperature was 35:25 °C day/night temperature.

Water sampling and analysis

Wastewater was analyzed before loading to each VUF-CW system, and its physicochemical characteristics are presented in Table 1. The wastewater was retained in the system for 3 days. Effluent grab samples were collected from the outlet of each treatment unit and analyzed immediately after sampling, without prior storage. This procedure was repeated four times, resulting in 16 replicate samples per treatment. At the end of each run, each VUF-CW unit was completely drained through the outlet before being refilled with fresh wastewater.

Table 1. Physicochemical characteristics of the diluted wastewater used in the experiment.

Note: The mean and standard error were calculated from 16 replicates in four repeated runs (four replicates per round)

The pH of the water samples was measured using a portable pH meter (Model: EZDO 6011, Taiwan), while the temperature (°C), electrical conductivity (EC), and total dissolved solids (TDS) were analyzed using a portable TDS and EC meter (Model: E-1 portable, China). Total suspended solids (TSS) were determined by the standard gravimetric method, with samples dried at 103–105 °C to constant weight. Dissolved oxygen (DO) and biochemical oxygen demand (BOD₅) were analyzed according to the standard method (APHA, 2017). Orthophosphate (PO₄^3--P) were analyzed according to the stannous standard methods (APHA, 2017). Ammonium (NH₄⁺-N) was determined by using a modified salicylate method (Quikchem Method no. 10-107-06-3B; Lachat Instruments, Milwaukee, WI, USA). Nitrate (NO₃^--N) was measured by the UV method (Oscarson et al., 1998).

Calculation and statistical analysis

The removal percentages of NH₄⁺-N, NO₃^--N, PO₄^3--P, and BOD₅ were calculated using the following formulas:

Removal (%) = [(C_i - C_e)/C_i] ×100

where C_i is influent concentration and C_e is effluent concentration

Statistical analyses were performed using SPSS Statistics version 17.0 (SPSS Inc., Chicago, IL, USA). Data were examined using one-way and two-way analysis of variance (ANOVA). Two-way ANOVA was used to evaluate the effects of two factors (planted x water recirculation) and their interaction. Within each planted and unplanted system, differences among water recirculation regimes were analyzed using one-way ANOVA followed by Duncan’s post hoc test. In addition, independent t-tests were performed to assess significant differences between planted and unplanted systems within each water recirculation regime. All statistical analyses were performed at a significance level of 0.05 (P < 0.05).

RESULTS

Improved DO concentrations by plant and water recirculation

The increase in effluent DO concentration in the VUF-CWs was affected by plantation and intermittent water recirculation (Table 2). Effluent DO concentrations were significantly higher in the systems planted with C. indica than in the unplanted systems (Table 4). In planted systems, effluent DO concentration significantly increased with increasing recirculation time, from 0 to 1.2, 3.4, and 3.9 mg L^-1 in the systems operating with no recirculation, 3:3 h, and 6:3 h recirculation/non-recirculation regime (Table 3, Figure 2A). Meanwhile, DO concentrations in unplanted systems ranged 0.6–2.0 mg L^-1 and also increased with increasing recirculation time (Table 3, Figure 2A).

Removal of BOD₅, nitrogen and phosphorus

Plantation and intermittent recirculation significantly affected the removal efficiencies of BOD₅, NH₄⁺-N, and NO₃^--N, whereas PO₄³⁻‑P removal was significantly affected only by plantation (Table 2). Under each water recirculation regime, planted systems achieved higher removal efficiency of BOD₅ and nitrogen than unplanted systems (Table 3 and 4). Increasing the recirculation time also improved the removal of BOD₅ and nitrogen in both the planted and unplanted systems. Approximately 73%, 73%, and 90% of BOD₅, NH₄⁺-N, and NO₃^--N were removed from wastewater in the systems planted with C. indica and operated with 6:3 h recirculation/non-recirculation regime. Meanwhile, unplanted systems without recirculation achieved BOD₅, NH₄⁺-N, and NO₃^--N removal efficiencies of approximately 8%, 26%, and 24%, respectively (Figure 2B–D). However, the removal efficiency of PO₄^3--P did not differ significantly among the treatments (Figure 2F). A slight increase in PO₄^3--P removal efficiency, from 12–15% in unplanted systems to 16–20% in planted systems, was observed.

Table 2. Two-way ANOVA (F-ratio) results of the effluent DO, removal efficiency of BOD₅, NH₄⁺-N, NO₃^--N, PO₄^3--P, TSS, and EC, and TDS increase in VUF-CWs (unplanted or planted with C. indica) operated under different intermittent recirculation regimes.

Note: *P < 0.05, **P < 0.01, ***P < 0.001

Table 3. Water quality analysis results (mean ± SE) of influent and effluent in VUF-CWs planted with C. indica and unplanted systems operated under different intermittent recirculation regimes.

Figure 2. Effluent dissolved oxygen (DO) concentration (A) and removal efficiencies of BOD₅ (B), NH₄⁺-N (C), NO₃⁻-N (D), TSS (E), and PO₄³⁻-P (F) in VUF-CWs planted with C. indica and in unplanted systems operated under different intermittent recirculation regimes. Different letters above the bars indicate significant differences among recirculation regimes within each planting treatment (planted or unplanted) (P < 0.05).

Table 4. Results of independent t-tests (P-values) comparing planted and unplanted treatments within each water recirculation regime.

Note: Bold values indicate significant differences at P < 0.05.

Water physical properties

Effluent pH, temperature, EC, TDS, and TSS were shown in Table 3.  Effluent pH slightly increased to 7.0–7.2, and the water temperature fluctuated between 28 and 32°C. TDS concentrations in the effluent also increased to 643–701 ppm after treatment, especially in planted systems. However, water recirculation regime did not show significant effects on TDS increasing percentage. There were no significant differences in water pH, temperature among the different systems. In contrast, EC in the effluent slightly decreased after treatment with both planted and unplanted systems and with/without water recirculation. TSS removal efficiencies were similar in the planted and unplanted systems (Table 4). However, increasing recirculation times resulted in lower TSS removal efficiencies in both planted and unplanted systems, whereas the highest TSS removal efficiency (approximately 91%) was observed in the no recirculation systems (Figure 2E).

DISCUSSION

In conventional VFCWs, oxygen generally diffuses from air at slow rates on the surface layer. Wetland plants can transfer oxygen from the atmosphere to the rhizosphere, serving as a major oxygen source in constructed wetland systems (Shukla et al., 2022). We found that DO concentrations in the planted systems were double those of the unplanted systems. This indicates that plant-derived oxygen was not limited to the rhizosphere and increased bulk-water DO via recirculation. Oxygen release from the roots of C. indica has been documented, as it can produce aerenchyma, particularly under hypoxia (Suriyakaew and Jampeetong, 2021) and plant-mediated oxygen transfer has also been suggested in constructed wetlands (Zorai et al., 2023). However, growing C. indica in CWs using biochar as a filter medium may influence plant growth, as found for Typha angustifolia L. (Janyasupab et al., 2023) and may also reduce aerenchyma formation and root porosity compared with gravel (Jampeetong and Janyasupab, 2022). To avoid these effects, biochar was added to the bottom layer of the system to filter suspended solids and organic matter. The negative effects of biochar were also prevented by the pumice in the middle layer. From the results, the roots of C. indica grew well and could supply more oxygen to the systems compared with the unplanted systems that obtained oxygen diffused from the atmosphere only. Increasing DO concentration supports aerobic microbial activities such as organic oxidation and nitrification (Zhou et al., 2020, Khajah et al., 2023). Hence, this study observed higher BOD₅ and NH₄⁺-N removal in planted systems than in unplanted systems, in response to higher DO. The higher NH₄⁺-N removal rates in planted systems could also be attributed to plants utilizing NH₄⁺-N for growth. Interestingly, NO₃^--N concentrations did not increase as a result of nitrification. Instead, high NO₃^--N removal was observed in the planted systems. Canna indica can assimilate both NH₄⁺-N and NO₃^--N and grow well in the presence of both N forms (Konnerup and Brix, 2010). This suggests that C. indica significantly contributes to the removal of NH₄⁺-N and NO₃^--N in VFCWs. Furthermore, the application of biochar may further promote nitrogen removal efficienct in VUF-CW systems. The cation exchange capacity (CEC) and porous structure of biochar, which can support NH₄⁺-N absorption, have been documented (Kharel et al., 2019; Janyasupab et al., 2023). Additionally, the high porosity of biochar may provide favorable habitats for microorganisms including nitrifying and denitrifying bacteria (Kizito et al., 2017; Dalahmeh et al., 2019). In the present study, microbial processes likely contributed to nitrogen removal in unplanted systems, where 25% and 24% of NH₄⁺-N and NO₃^--N were removed, respectively. Furthermore, biochar has been reported to enhance denitrification by facilitating electron transfer (Tong et al., 2022). Fu et al. (2020) also reported that aerobic denitrifying bacteria could contribute to NO₃^--N removal in CWs. However, microbial abundance and community composition were not investigated in the present study. Future studies incorporating microbial community analysis would be valuable to verify the mechanisms for the enhanced nitrogen removal in biochar-based VUF-CWs. Higher removal efficiencies of NH₄⁺-N and NO₃^--N (64% and 43%, respectively) were observed in the planted systems without recirculation, where more oxygen was supplied by plants. Similarly, in tidal-flow constructed wetlands planted with Colocasia esculenta (L.) Schott. and amended with biochar, NH₄⁺-N and NO₃^--N removal efficiencies reached 88.16 % and 57.85 %, respectively, which were higher than in systems without biochar or without plant (Chand et al., 2022). The present study clearly indicates that C. indica plays an important role in enhancing oxygen availability (higher effluent DO), supporting aerobic biodegradation and nitrification, while contributing to N removal via plant uptake. In addition, the use of biochar and pumice could provide habitats for microorganisms, thereby enhancing BOD₅ and nitrogen removal.

Setting up water recirculation systems is another strategy that can promote oxygen distribution from root zone to deeper water layers in VFCW, and oxygen supply can be enhanced in highly porous substrates such as biochar and pumice (Jampeetong and Janyasupab, 2022; Shukla et al., 2022). In this study, the DO concentration increased with increasing recirculation time, reaching 3.9 mg L^-1 in the planted system. A significant decrease in BOD₅, NH₄⁺-N, and NO₃^--N concentrations with increasing recirculation time was also observed in both planted and unplanted systems. In previous studies, high BOD₅ removal efficiency was also observed in recirculated VFCWs planted with Phragmites australis (Cav.) Trin. ex Steud. (Lavrova and Koumanova, 2010). Likewise, improved NH₄⁺-N removal was observed in intermittently recirculated VFCWs (Foladori et al., 2013). In addition, approximately 99% removal of both BOD₅ and NH₄⁺-N was achieved in VFCWs planted with P. australis and Iris pseudacorus L. and operated with and without effluent recycling (Arias et al., 2022). Compared with the systems without water recirculation, effluent DO concentrations were low, particularly in the unplanted systems (0.6 mg L^-1), which was consistent with the low NH₄⁺-N removal observed. Chang et al. (2012) reported that saturated wetland beds of integrated vertical down-flow and up-flow constructed wetlands had low effluent DO concentration (0.81–0.85 mg L^-1), which restricted nitrification and resulted in low total nitrogen (TN) removal efficiencies of 12.8–15.0%. The findings of the present study indicate that intermittent recirculation may create alternating high- and low-oxygen conditions that promote coupled nitrification–denitrification and enhancing nitrogen transformation. The reduction in NH₄⁺-N may be partly associated with enhanced nitrification in oxygenated zones, where NH₄⁺ is oxidized to NO₂^- and subsequently to NO₃^- (Xia et al., 2020). Meanwhile, the decline in NO₃^--N suggests that denitrification may also have occurred within the system during non-recirculation periods, when oxygen availability was lower. In partially saturated vertical flow CWs wetlands, dissolved oxygen can be consumed by microbial and root respiration, leading to low-oxygen conditions that favor denitrifying activity (Xia et al., 2020; Zhou et al., 2020). Furthermore, oxygen gradients within biofilms and porous media may create localized oxic and anoxic microenvironments that facilitate simultaneous nitrification and denitrification (SND). The reduction in BOD₅ also suggests active microbial degradation of organic matter within the system, which may enhance nitrogen transformation processes. However, because NO₂^--N, total nitrogen (TN) and organic nitrogen were not measured, the relative contributions of nitrification, denitrification, plant uptake, and biochar adsorption processes could not be quantified and future studies on nitrogen mass balance and microbial analyses should be conducted to verify the dominant nitrogen removal pathways. Furthermore, water recirculation could enhance plant nitrogen uptake resulting in the highest removal efficiencies of NH₄⁺-N (73%), and NO₃^--N (90%) in the planted system operated under the 6:3 h recirculation/non-recirculation regime. With water recirculation, nutrients are more evenly distributed throughout the root zones, thereby increasing their contact with plant roots. Under well-oxygenated conditions, high DO can also enhance root nitrogen uptake (NH₄⁺-N and NO₃^--N) (Manokieng and Jampeetong, 2025). Hence, intermittent recirculation could play key roles in enhanced plant nitrogen uptake and support healthy root growth in this study, while intermittent operation can improve oxygen availability for microbial activities.

Regarding PO₄^3--P removal, it had relatively low removal efficiency of approximately 12–20% compared to removal of NH₄⁺-N and NO₃^--N. This suggests that phosphorus removal mechanisms were limited under experimental conditions. This could be a result of the low sorption capacity of filtration materials (Vymazal, 2010). He et al. (2022) reported that unmodified biochar had a limited capacity to adsorb phosphate. Most biochar surfaces have a negative charge and adsorb very little PO₄^3-. Hence, phosphate adsorption onto the longan biochar may be limited in this study. In addition, phosphorus can also be removed by plant uptake. Chand et al. (2022) reported approximately 20% PO₄^3- removal in an unplanted tidal-flow constructed wetlands (TFCWs) containing biochar (mixed with sand in the top layer), but observed an increase in PO₄^3- removal to approximately 58 % in the system planted with C. esculenta (with biochar), after the first 3-day tidal-flow cycle. In our study, PO₄^3--P removal was only slightly higher in the planted than unplanted systems and intermittent recirculation had no significant effect. This suggests that although C. indica is capable of phosphorus uptake, plant assimilation alone is often insufficient to achieve substantial phosphorus removal. Furthermore, magnesium (Mg²⁺) may be released from biochar via cation exchange with NH₄⁺-N. Under favorable pH conditions, particularly at moderately alkaline pH, Mg²⁺ can precipitate with phosphate to form Mg–P compounds (He et al., 2022). However, in the present study, the effluent pH remained nearly neutral (7.0–7.2), which may be less favorable for phosphate precipitation. This may partly explain the relatively low PO₄^3--P removal observed.

TSS removal was enhanced by the specific surface area (SSA) and pore volume (PV) of biochar (Janyasupab and Jampeetong, 2022). A study by de Rozari et al. (2015) found that TSS was better removed in systems with biochar-amended sand than those with pure sand, particularly when septage was loaded. The present study showed that TSS removal was effective in both planted and unplanted systems under no-recirculation conditions, with removals of 90–91%. However, lower TSS removal was observed under prolonged water recirculation and was likely associated with increased hydraulic disturbance and resuspension of retained particulates within the substrate matrix. Based on the recirculation flow rate (2.5 L min^-1) and system cross-sectional area (19.5 x 19.5 cm²), the superficial flow velocity was approximately 6.6 cm min^-1, corresponding to an estimated interstitial velocity (pore-water velocity) of approximately 14 cm min^-1. Prolonged operation at this recirculation rate may have increased shear forces within the porous media and promoted resuspention of the retained particles, thus reducing TSS removal efficiency. Future design improvements may include lower recirculation rates, improved flow distribution, or incorporation of settling zones to minimize resuspension of solids in the system.

Using biochar as a filter medium can release readily soluble base cations (e.g., Ca²⁺, Mg²⁺, K⁺, Na⁺) from ash/salts into the surrounding solution (Munera-Echeverri et al., 2018), This release of dissolved ions could contribute to increased TDS. A study by Janyasupab et al. (2023) showed that effluent TDS concentration increased (12–19%) in the longan biochar-based systems, which is comparable to our study (12–36% of TDS increase). Since the same quantity of biochar was used and cleaned several times before use, there was a slight increase in TDS without significant differences in TDS concentrations under different water recirculation regimes. Generally, EC correlates with TDS. However, in this study, EC slightly decreased by less than 10%. Decreasing EC suggested a reduction in ion concentrations in water, which was associated with the removal of NH₄⁺-N and NO₃^--N. The decrease in EC was not as high as the decrease in NH₄⁺-N and NO₃^--N. This indicated the release of other ions from the substrates that maintained the EC level and contributed to an increase in TDS. TDS represents all solids dissolved in water, including both ionic and non-ionic substances; hence, the increase in TDS in this phenomenon was attributed to the dissolution of non-ionic compounds, such as organic materials from biodegradation processes.

CONCLUSION

From the study, C. indica planted VUF-CWs significantly increased DO and nutrient removal, particularly nitrogen, while PO₄^3--P was moderately removed (20%). Operating the system with prolonged intermittent recirculation improved dissolved oxygen levels and promoted organic degradation, resulting in high BOD₅, NH₄⁺-N, and NO₃^--N removal efficiencies. The use of biochar and pumice as filter media enhances these removal processes by providing large surface areas for cation exchange and microbial attachment, also enhancing oxygen preservation. However, extended recirculation time was found to reduce TSS removal due to hydraulic disturbances. These lab-scale findings demonstrate that integrating intermittent circulation with longan biochar-pumice media and C. indica can improve the performance of VUF-CWs through enhanced oxygen transfer and nitrogen removal. The findings provided a basis for the development of more sustainable and cost-effective constructed wetland systems. However, full-scale studies are needed to evaluate operational feasibility and long-term stability such as biochar performance, organic matter accumulation, clogging potential, and long-term treatment efficiency under practical conditions.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University (Mae Hia Campus), Chiang Mai, Thailand, for providing the wastewater used in this study. The authors also thank Dr. Pakawat Janyasupab for his assistance with wastewater collection and analysis, and Mr. Tanapat Teparak for his contributions to the design and construction of the VFCW model.

AUTHOR CONTRIBUTIONS

Kalanyoo Thompradit: Investigation (Lead), Data Curation (Lead), Formal Analysis (Lead), Writing-Original Draft (Equal); Tararag Pincum: Formal Analysis (Equal), Writing- Reviewing and Editing (Equal); Arunothai Jampeetong: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Equal), Project Administration (Lead), Writing- Reviewing and Editing (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

During the preparation of this manuscript, the authors used Matthew AI (Chiang Mai University's generative AI platform, based on the GPT-5.2 model) solely for English language editing, including grammar checking and language polishing. The tool was not used to generate, analyze, or interpret any research content. All substantive content, interpretations, and conclusions are entirely the authors' own. The authors reviewed and verified all AI-assisted edits and take full responsibility for the content of the final manuscript.

REFERENCES

American Public Health Association, American Water Works Association, Water Environment Federation (APHA-AWWA-WEF). 2017. Standard Methods for the Examination of Water and Wastewater, 23^rd ed. Washington DC: American Public Health Association.

Amorim, C.A. and Moura, A.N. 2021. Ecological impacts of freshwater algal blooms on water quality, plankton biodiversity, structure, and ecosystem functioning. Science of the Total Environment. 758: 143605. https://doi.org/10.1016/j.scitotenv.2020.143605

Arias, C.A., Istenic, D., Stein, O., Zhai, X., Kilian, R., Vera-Puerto, I., and Brix, H. 2022. Effects of effluent recycle on treatment performance in a vertical flow constructed wetland. Ecological Engineering. 180: 106675. https://doi.org/10.1016/j.ecoleng.2022.106675

Baggiotto, C., Decezaro, S.T., Lutterbeck, C.A., Friedrich, M., González Ramírez, R.J.M., and Wolff, D.B. 2025. Nitrogen removal in vertical flow constructed wetlands: The influence of recirculation and partial saturation. Ecological Engineering. 212: 107519. https://doi.org/10.1016/j.ecoleng.2025.107519

Chand, N., Kumar, K., and Suthar, S. 2022. Enhanced wastewater nutrients removal in vertical subsurface flow constructed wetland: Effect of biochar addition and tidal flow operation. Chemosphere. 286: 131742. https://doi.org/10.1016/j.chemosphere.2021.131742

Chang, J.J., Wu, S.Q., Dai, Y.R., Liang, W., and Wu, Z.B. 2012. Treatment performance of integrated vertical-flow constructed wetland plots for domestic wastewater. Ecological Engineering. 44: 152-159. https://doi.org/10.1016/j.ecoleng.2012.03.019

Cui, L., Ouyang, Y., Lou, Q., Yang, F., Chen, Y., Zhu, W., and Luo, S. 2010. Removal of nutrients from wastewater with Canna indica L. under different vertical-flow constructed wetland conditions. Ecological Engineering. 36(8): 1083-1088. https://doi.org/10.1016/j.ecoleng.2010.04.026

Dalahmeh, S.S., Assayed, A., and Stenström, Y. 2019. Combined vertical-horizontal flow biochar filter for onsite wastewater treatment—Removal of organic matter, nitrogen and pathogens. Applied Sciences. 9(24): 5386. https://doi.org/10.3390/app9245386

de Rozari, P., Greenway, M., and El Hanandeh, A. 2015. An investigation into the effectiveness of sand media amended with biochar to remove BOD₅, suspended solids and coliforms using wetland mesocosms. Water Science & Technology. 71(10): 1536-1544. https://doi.org/10.2166/wst.2015.120

Foladori, P., Ruaben, J., and Ortigara, A.R.C. 2013. Recirculation or artificial aeration in vertical flow constructed wetlands: A comparative study for treating high load wastewater. Bioresource Technology. 149: 398-405. https://doi.org/10.1016/j.biortech.2013.09.099

Fu, G., Wu, J., Han, J., Zhao, L., Chan, G., and Leong, K. 2020. Effects of substrate type on denitrification efficiency and microbial community structure in constructed wetlands. Bioresource Technology. 307: 123222. https://doi.org/10.1016/j.biortech.2020.123222

He Q., Li, X. and Ren, Y. 2022. Analysis of the simultaneous adsorption mechanism of ammonium and phosphate on magnesium‑modified biochar and the slow release effect of fertilizer. Biochar. 4: 25. https://doi.org/10.1007/s42773-022-00150-5

Jampeetong, A. and Janyasupab, P. 2022. Effects of substrate types on nitrogen removal efficacy and growth of Canna indica L. Applied Environmental Research. 44(1): 42-53. https://doi.org/10.35762/AER.2021.44.1.4

Janyasupab, P. and Jampeetong, A. 2022. Performance of porous substrates for domestic wastewater treatment under prolonged hydraulic retention time. Applied Environmental Research. 44(3): 45-58. https://doi.org/10.35762/AER.2022.44.3.4

Janyasupab, P., Brix, H., and Jampeetong, A. 2023. Effects of longan biochar as filter materials on plant responses and wastewater treatment by Typha angustifolia L. Natural and Life Sciences Communications. 22(3): e2023035. https://doi.org/10.12982/NLSC.2023.035

John, Y., Langergraber, G., Adyel, T.M., and David, V.E., Jr. 2020. Aeration intensity simulation in a saturated vertical up-flow constructed wetland. Science of The Total Environment. 708: 134793. https://doi.org/10.1016/j.scitotenv.2019.134793

Khajah, M., Bydalek, F., Babatunde, A.O., Al-Matouq, A., Wenk, J., and Webster, G. 2023. Nitrogen removal performance and bacterial community analysis of a multistage step-feeding tidal flow constructed wetland. Frontiers in Water. 5: 1128901. https://doi.org/10.3389/frwa.2023.1128901

Kharel, G., Sacko, O., Feng, X., Morris, R.J., Phillips, C.L., Trippe, K., Kumar, S., and Lee, W.J. 2019. Biochar surface oxygenation by ozonization for super high cation exchange capacity. ACS Sustainable Chemistry & Engineering. 7(19): 16410-16418. https://doi.org/10.1021/acssuschemeng.9b03536

Kizito, S., Lv, T., Wu, S., Ajmal, Z., Luo, H., and Dong, R. 2017. Treatment of anaerobic digested effluent in biochar-packed vertical flow constructed wetland columns: Role of media and tidal operation. Science of the Total Environment. 592: 197-205. https://doi.org/10.1016/j.scitotenv.2017.03.125

Konnerup, D. and Brix, H. 2010. Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates. Aquatic Botany. 92(2): 142-148. https://doi.org/10.1016/j.aquabot.2009.11.004

Lavrova, S. and Koumanova, B. 2010. Influence of recirculation in a lab-scale vertical flow constructed wetland on the treatment efficiency of landfill leachate. Bioresource Technology. 101: 1756-1761. https://doi.org/10.1016/j.biortech.2009.10.028

Manokieng, M. and Jampeetong, A. 2025. Growth, morphology, and nitrogen uptake adaptivity of Phragmites karka in response to nitrogen forms and oxygen availability. Aquatic Botany. 201: 103918. https://doi.org/10.1016/j.aquabot.2025.103918

Munera-Echeverri, J.L., Martinsen, V., Strand, L.T., Zivanovic, V., Cornelissen, G., and Mulder, J. 2018. Cation exchange capacity of biochar: An urgent method modification. Science of the Total Environment. 642: 190-197. https://doi.org/10.1016/j.scitotenv.2018.06.017

Oscarson, P., Ingemarsson, B., Ugglas, M.A., and Larsson, C.M. 1988. Characteristics of NO₃^- uptake in Lemna and Pisum. Plant and Soil. 111: 203-205. https://doi.org/10.1007/BF02139939

Phewnil, O., Chunkao, K., Prabhuddham, P., and Pattamapitoon, T. 2024. Application of different aquatic plants in an alternated fill and drain wetland system of Phetchaburi municipal wastewater treatment in Thailand. Environmental Science and Pollution Research. 31: 1304-1313. https://doi.org/10.1007/s11356-023-31266-1

Pinninti, R., Kasi, V., Sallangi, L.K.S.V.P., Landa, S.R., Rathinasamy, M., Sangamreddi, C., and Dandu Radha, P.R. 2022. Performance of Canna indica based microscale vertical flow constructed wetland under tropical conditions for domestic wastewater treatment. International Journal of Phytoremediation. 24(7): 684-694. https://doi.org/10.1080/15226514.2021.1962800

Pucher, B. and Langergraber, G. 2019. The state of the art of clogging in vertical flow wetlands. Water. 11(11): 2400. https://doi.org/10.3390/w11112400

Rout, P.R., Shahid, M.K, Dash, R.R., Bhunia, P., Liu, D., Varjani, S., Zhang, T.C., and Surampalli, R.Y. 2021. Nutrient removal from domestic wastewater: A comprehensive review on conventional and advanced technologies. Journal of Environmental Management. 296: 113246. https://doi.org/10.1016/j.jenvman.2021.113246

Santos, J., Rodrigues, S., Magalhães, M., Rodrigues, K., Pereir, L., and Marinho, G. 2024. A state-of-the-art review (2019-2023) on constructed wetlands for greywater treatment and reuse. Environmental Challenges. 16: 100973. https://doi.org/10.1016/j.envc.2024.100973

Sharma, M., Sharma, N.R., and Kanwar, R.S. 2024. Performance analysis of mesocosm-constructed wetland containing agricultural waste-derived substrates for treatment of wastewater. Environmental Monitoring and Assessment. 196: 1220. https://doi.org/10.1007/s10661-024-13411-6

Shukla, A., Parde, D., Gupta, V., Vijay, R., and Kumar, R. 2022. A review on effective design processes of constructed wetlands. International Journal of Environmental Science and Technology. 19: 12749-12774. https://doi.org/10.1007/s13762-021-03549-y

Soundaranayaki, K. and Gandhimathi, R. 2019. Performance of various media in vertical flow constructed wetland for the treatment of domestic wastewater. Desalination and Water Treatment. 146: 57-67. https://doi.org/10.5004/dwt.2019.23566

Suriyakaew, T. and Jampeetong, A. 2021. Effects of dissolved O₂ and Fe availability on growth, morphology, aerenchyma formation and radial oxygen loss of Canna indica L. and Heliconia psittacorum L.f. Chiang Mai University Journal of Natural Sciences. 20(4): e2021086. https://doi.org/10.12982/CMUJNS.2021.086

Thalla, A.K., Devatha, C.P., Anagh, K., and Sony, E. 2019. Performance evaluation of horizontal and vertical flow constructed wetlands as tertiary treatment option for secondary effluents. Applied Water Science. 9: 147. https://doi.org/10.1007/s13201-019-1014-9

Tong, H., Duan, Z., Yang, X., Li, E., Liu, Y., Zhou, H., Zhang, X., Yang, X., and Xu, W. 2022. Characteristics of sludge-based pyrolysis biochar and its application of enhancing denitrification. Chemosphere. 309: 136813. https://doi.org/10.1016/j.chemosphere.2022.136813

Vymazal, J. 2010. Constructed wetlands for wastewater treatment. Water. 2(3): 530−549. https://doi.org/10.3390/w2030530

Vymazal, J. 2022. The Historical development of constructed wetlands for wastewater treatment. Land. 11: 174. https://doi.org/10.3390/land11020174

Xia, Z., Liu, G., She, Z., Gao, M., Zhao, Y., Guo, L., and Jin, C. 2020. Performance and bacterial communities in unsaturated and saturated zones of a vertical-flow constructed wetland with continuous-feed. Bioresource Technology. 315: 123859. https://doi.org/10.1016/j.biortech.2020.123859

Xiao, J., Lin, G., Cao, Z., Chu, S., Cui, L., Yang, Y., and Wu, X. 2023. A shallow constructed wetland combining porous filter material and Rotala rotundifolia for advanced treatment of municipal sewage at low HRT. Environmental Science and Pollution Research. 30: 27593-27602. https://doi.org/10.1007/s11356-022-24111-4

Zhao, Y.J., Hui, Z., Chao, X., Nie, E., Li, H.J., He, J., and Zheng, Z. 2011. Efficiency of two-stage combinations of subsurface vertical down-flow and up-flow constructed wetland systems for treating variation in influent C/N ratios of domestic wastewater. Ecological Engineering. 37: 1546-1554. https://doi.org/10.1016/j.ecoleng.2011.06.005

Zhou, L., Wang, J., Xu, D., Li, Y., Yao, B., and Howard, A. 2020. Responses of nitrogen transformation and dissolved oxygen in constructed wetland to biochar and earthworm amendment. Environmental Science and Pollution Research. 27: 29475-29484. https://doi.org/10.1007/s11356-020-09274-2

Zorai, A., Benzahi, K., Labed, B., Ouakouak, A., Serraoui, M., and Bouhoreira, A. 2023. Performance of Canna indica and Typha latifolia in mono and mixed culture for secondary wastewater treatment in constructed wetlands with vertical flows under arid conditions (Touggourt, Algeria). Water Practice & Technology. 18(1): 53-67. https://doi.org/10.2166/wpt.2022.163         

OPEN access freely available online

Natural and Life Sciences Communications

Chiang Mai University, Thailand. https://cmuj.cmu.ac.th