Influence of operational conditions on the efficiency of Wolffia brasiliensis for polishing domestic wastewater of facultative stabilization ponds

Influence of operational conditions on the efficiency of Wolffia brasiliensis for polishing domestic wastewater of facultative stabilization ponds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of operational conditions on the efficiency of Wolffia brasiliensis for polishing domestic wastewater of facultative stabilization ponds Ana Beatriz Laluce Vaz, Davi Gasparini Fernandes Cunha, Gleyson B. Castro, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6099077/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Stabilization ponds are widely used for wastewater treatment in developing countries but have limitations in removing certain pollutants, necessitating polishing processes to enhance effluent quality and mitigate environmental impacts. Aquatic macrophytes, particularly Lemnaceae , offer a low-cost, efficient alternative due to their rapid reproduction and high nutrient absorption. This study evaluated the effects of operational conditions on Wolffia brasiliensis development and its efficiency in polishing wastewater from facultative stabilization ponds. The parameters assessed were chemical oxygen demand (COD), biochemical oxygen demand (BOD₅,₂₀), total nitrogen (TN), and total phosphorus (TP). Three cultivation conditions were tested: untreated effluent, recirculated effluent (6 × 10⁻³ L s⁻¹ flow rate), and 10% diluted effluent. Experiments were conducted indoors (21,818 lux, 24°C ± 1.3°C) and outdoors (natural conditions, 31°C ± 3.5°C). Biofilm formation hindered macrophyte growth and contaminant removal in outdoor units. Indoor conditions yielded higher biomass (85.87 ± 11.9 g m⁻² d⁻¹) than outdoor ones (31.35 ± 9.3 g m⁻² d⁻¹). Recirculated effluent led to the highest growth rates (42.06 ± 33.1 g m⁻² d⁻¹ indoor, 15.70 ± 11.7 g m⁻² d⁻¹ outdoor). All W. brasiliensis units significantly improved pollutant removal compared to controls. These findings highlight W. brasiliensis as an effective, sustainable solution for polishing effluents from facultative stabilization ponds, particularly for BOD₅,₂₀, COD, TN, and TP removal, reinforcing its potential for wastewater management in developing countries. Wastewater treatment Organic matter removal Nutrient removal Lemnaceae Macrophyte Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Stabilization ponds are among the simplest methods for wastewater treatment because they are characterized by low costs of implementation, maintenance, and operation (Arthur 1983; Von Sperling 2002). Such systems are particularly well-suited for applications in developing countries from tropical and subtropical regions, where land areas are generally available at relatively low prices and climatic conditions such as high temperatures and year-round solar radiation favor the biodegradation process (Arthur 1983; Mahapatra et al. 2022). According to the Brazilian Water Agency (ANA), in 2020, stabilization ponds accounted for 32% of the 3,600 sewage treatment plants (STPs) under operation in Brazil, serving as the primary treatment technology (ANA 2020). However, while such ponds promote a satisfactory removal of organic matter, their efficiency for nutrient removal, mainly regarding nitrogen and phosphorus, is limited (Fujioka et al. 2020), resulting in the eutrophication of receiving water bodies and overall water quality decrease (Miwa et al. 2007). Hence, effluent polishing technologies that offer both high efficiency and low installation costs are often required and have seen increasing adoption. Aquatic macrophytes have emerged as a prominent strategy for nutrient and organic matter abatement from wastewater, being recognized as a versatile and scalable intervention that can be adapted to diverse regional and operational contexts (Herzog and Rozado 2010; Uysal 2013). Among aquatic macrophyte species, floating plants of the Lemnaceae family are particularly notable for their simple structural organization and predominance of photosynthetic tissues (Wolverton and McDonald 1980; Ziegler et al. 2016; Chakrabarti et al. 2018). They are tolerant to environmental pollutants, including metals (Cheng and Stomp 2009; Ziegler et al. 2016), and show potential for removing suspended solids and biochemical oxygen demand (Mohedano et al. 2014). Due to such advantageous traits, Lemnaceae species have been increasingly employed in wastewater treatments and post-treatment processes (Ziegler et al. 2017; Toyama et al. 2018). Macrophytes of the genus Wolffia - recognized as the smallest macrophytes in Lemnaceae - are distinguished by their rapid reproduction and growth rates (Suppadit et al. 2008). Kumar et al. (2022) evaluated the nutrient removal capacity of Wolffia globosa under controlled laboratory conditions and reported an above 99% efficiency for removing total inorganic nitrogen and orthophosphate. Soda et al. (2013) evaluated the nutrient removal capacity of Wolffia arrhiza under controlled laboratory mesocosms supplied with synthetic wastewater and the findings revealed nitrogen and phosphorus removal rates of 0.03–0.18 mg N m⁻² d⁻¹ and 0.023–0.079 mg P m⁻² d⁻¹, respectively. Similarly, Zhang et al. (2011) investigated the potential of W. arrhiza for polishing effluents from stabilization ponds, reporting removal efficiencies of 84% for total phosphorus (TP), 46% for total nitrogen (TN), 76% for biochemical oxygen demand (BOD₅,20), and 69% for chemical oxygen demand (COD). Numerous studies have underscored the effectiveness of Lemnaceae species in wastewater treatment, particularly in nutrient removal. The most extensively studied genera include Lemna (Valderrama et al. 2002; Uysal 2013; Chen et al. 2018; Toyama et al. 2018), Landoltia (Garcia 2015; Chen et al. 2018; Toyama et al. 2018; Zanetoni Filho 2019; Cerqueira 2021), and Spirodela (Cheng and Stomp 2009; Chen et al. 2018; Toyama et al. 2018). In contrast, research on the genus Wolffia remains relatively scarce (Landolt and Kandeler 1987; Kotowska et al. 2018). To date, most investigations on Wolffia have been conducted in continental (Kotowska et al. 2018) and temperate climates (Zhang et al. 2011; Soda et al. 2013; Kumar et al. 2022), with limited data available on its performance under tropical climatic conditions. This study evaluated the polishing process of previously treated wastewater from a Wastewater Treatment Plant (WWTP) located in Ilha Solteira (São Paulo State, Brazil) using W. brasiliensis macrophyte. Three distinct operational conditions, namely, undiluted effluent without recirculation (CW), effluent diluted to 10% without recirculation (ED), and undiluted effluent with recirculation (CR) facilitated by a submersible pump were assessed. The study focused on the influence of operational conditions on the relative growth rate of the macrophyte and the removal efficiency of key contaminants, including chemical oxygen demand (COD), biochemical oxygen demand (BOD 5,20 ), total nitrogen (TN), and total phosphorus (TP). The findings offer valuable insights for the design and construction of wetlands, supporting the development of more sustainable solutions in environmental management. 2. Materials and Methods The study was conducted at the Sanitation Laboratory of the Civil Engineering Department (20°25'42.4" S 51°20'29.9" W), at São Paulo State University “Júlio de Mesquita Filho” (UNESP), Ilha Solteira campus, São Paulo, Brazil. 2.1. Characteristics of the studied wastewater The effluent utilized was collected from the outlet of a primary facultative pond at the Ilha Solteira Wastewater Treatment Plant (WWTP), which includes a preliminary treatment system comprising a screening unit and a sand trap, followed by two primary facultative ponds of 458 m length, 105 m width, and 1.5 m depth (Costa 2015). The initial concentrations of chemical oxygen demand (COD), biochemical oxygen demand (BOD 5,20 ), total nitrogen (TN), total phosphorus (TP), turbidity, total solids (TS), total suspended solids (TSS), and dissolved solids (DS) in the effluent were analyzed according to APHA (2017) methods (Table 1 ). Table 1 Initial characterization of the studied effluent used in the experimental polishing units Parameters Mean ± SD COD (mg L − 1 ) 371.00 ± 1.53 BOD 5,20 (mg L − 1 ) 73.94 ± 0.93 TN (mg L − 1 N) 7.05 ± 0.05 TP (mg L − 1 P) 3.26 ± 0.17 Turbidity (NTU) 220.00 ± 1.73 TS (mg L − 1 ) 680.00 ± 15.58 TSS (mg L − 1 ) 170.00 ± 11.00 DS (mg L − 1 ) 510.00 ± 5.00 Note. SD: Standard deviation obtained by three analytical replications. COD: Chemical Oxygen Demand; BOD 5,20 : Biochemical Oxygen Demand; TN: Total Nitrogen; TP: Total Phosphorus; TS: Total Solids; TSS: Total Suspended Solids; DS: Dissolved Solids. 2.2. Plant material, microscopic analysis and species identification The macrophyte was collected from the Wastewater Treatment Plant (WWTP) at the Provisional Detention Center of São José do Rio Preto (20°41'17" S 49°20'17" W), São Paulo, Brazil. The WWTP comprises a primary anaerobic pond, a secondary facultative pond, and two maturation ponds in series. Wolffia was found to develop spontaneously at all stages of the treatment process; however, the specimens used in this study were collected from the secondary facultative pond. Following collection, the macrophyte was cultivated and maintained in a 200 L polyethylene tank in an outdoor area adjacent to the laboratory. The tank received natural light and was supplied with the domestic wastewater used in the experimental trials. An analysis under a binocular optical microscope (50x magnification) confirmed the macrophyte species selected, whose morphological characteristics, including presence of blackened cells and occurrence of papillae on the upper surface of the plant, were observed. According to Landolt (1994), W. brasiliensis is distinguished from other species by the presence of darkened cells and fronds of 0.3 to 1.4 mm length. Such features were identified in the specimens used in this study (Fig. 1 ). 2.3. Experimental design The assessment of the growth potential of the macrophyte under varying conditions involved analyses of three distinct cultivation scenarios, namely, undiluted effluent without recirculation (CW), effluent diluted to 10% without recirculation (ED), and undiluted effluent with recirculation facilitated by a 2.5 W submersible pump operating at 6 × 10⁻³ L s⁻¹ continuous flow rate (CR). Additionally, control units containing only effluent (C) were included for evaluations of the self-purification capacity of the effluent and the natural decay of the studied parameters (Fig. 2 ). The experiment was conducted in two distinct environments. In an indoor one, 12 experimental polyethylene units containing 3 L of effluent each were arranged randomly within a wooden box (Fig. 2 ). The setup was illuminated by two tubular LED lamps (3000 K, 18 W, and 1.20 m length) connected to an analog photoperiod timer set to a 12 h light: 12 h dark cycle. The second environment was an outdoor area adjacent to the laboratory, where 12 experimental units were randomly arranged within a greenhouse of 3 m width, 3 m length, and 2.78 m height. The initial density of fresh Wolffia biomass in all experimental units was standardized at 200 g m⁻². The volume of evaporated effluent in each unit was replenished weekly towards maintaining the experimental conditions. The evaporated volume was systematically monitored and recorded, with corrections applied for accurate calculations of the removal efficiencies of the evaluated parameters. 2.4. Growth rate Wolffia growth was quantified in each experimental unit with the use of Relative Growth Rate (RGR) (g m⁻² d⁻¹) based on dry matter, as described by Verma and Suthar (2014), and calculated by Eq. 1. $$\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:RGR=\frac{DM}{N.A}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\left[1\right]$$ where DM = dry matter during the growing season (g), N = number of growing days, and A = surface area of ​​the growing section (m²). 2.5. Monitoring parameters and statistical analysis The water quality parameters used for evaluations of the efficiency of the effluent polishing process, namely biochemical oxygen demand (BOD 5,20 ), total phosphorus (TP), total nitrogen (TN), and chemical oxygen demand (COD), were analyzed weekly over a 35-day period, according to the Standard Methods for the Examination of Water and Wastewater (APHA 2017). The ambient temperature was monitored daily by two digital thermohygrometers that recorded maximum and minimum values, while pH was measured weekly by a portable digital pHmeter from Hanna Instruments, following the guidelines provided by APHA (2017). The determination of the reduction efficiencies of BOD 5,20 , COD, TP, and TN was based on the quantification limits specified by APHA (2017). A data analysis was conducted using a completely randomized design, with factor A representing type of treatment, factor B representing the environment (internal or external), and time as a subdivided plot. A statistical analysis was performed with the use of Sisvar software, applying analysis of variance (ANOVA) and Scott-Knott mean clustering test, both with a 5% significance level. The approach enables assessing the significance of the environment (internal versus external) and the growing conditions within each environment (diluted effluent, undiluted effluent, and effluent with recirculation). All the graphs used in this work were generated using Origin® 2023 software. 3. Results and discussion 3.1 Monitoring parameters The external temperature was significantly higher (p < 0.05) compared to the internal one, with averages of 24°C indoor and 31°C outdoor (Fig. 3 ). A greater standard deviation was observed in external temperature, which can be attributed to the natural variability of outdoor environments, The optimal temperature range for Wolffia cultivation is between 17°C-30°C and 17.5°C-34°C according to Skillicorn et al. (1993) and Hasan and Chakrabarti (2009), respectively. The indoor environment showed higher growth rates for W. brasiliensis compared to the outdoor one, which frequently exceeded the ideal temperature range outlined by Skillicorn et al. (1993) and Hasan and Chakrabarti (2009). Kumar et al. (2022) and Chakrabarti et al. (2018) successfully cultivated W. globosa under controlled laboratory conditions at 31°C using synthetic effluent and Lemna minor under outdoor conditions at 25–28°C, respectively, both similar to the conditions observed in this study. Overall, regarding the pH parameter, no significant differences were detected among operation conditions within the same environment on the 7th, 21st, and 28th days (Fig. 4 ). However, experimental units in outdoor environments showed significantly (p < 0.05) higher pH values (9.81 ± 1.3) compared to indoor ones (7.83 ± 0.4) from the 7th day of the experiment (Fig. 4 ), highlighting the influence of environmental factors such as temperature and light on pH levels. Skillicorn et al. (1993) defined the optimal pH range for Wolffia development as 7.00–8.00, while Hasan and Chakrabarti (2009) reported that Lemnaceae species can tolerate pH levels between 5 and 9. Thus, the pH values recorded under indoor laboratory conditions were considered viable and associated with higher macrophyte growth rates. In contrast, lower growth rates coincided with high outdoor temperatures and elevated pH levels. The indoor pH values in this study align with those reported by Kotowska et al. (2018) for W. arrhiza (7.60–7.90), which supported satisfactory growth, and with those found by Sirirustananun and Jongput (2021) (7.50–7.79) for the same species. Kumar et al. (2022) observed optimal pH values when cultivating W. globosa but reported an increase to 10.3 by the end of the culture, a trend consistent with the present study's findings for outdoor experimental units. 3.2 Relative growth rate In both environments, CR showed significantly higher biomass and growth rate values (p < 0.05) compared to CW and ED (Fig. 5 ), which can be attributed to the recirculation of nutrients within the system. Conversely, ED showed significantly lower values (p < 0.05) relative to CW and CR, due to reduced nutrient concentrations caused by the dilution process. The maximum relative growth rates for all experimental units were recorded on the 7th day of cultivation, i.e., 85.0 g m⁻² d⁻¹ for indoor CW unit, 32.38 g m⁻² d⁻¹ for outdoor CW unit, 74.04 g m⁻² d⁻¹ for indoor ED unit, and 26.19 g m⁻² d⁻¹ for outdoor ED unit, as well as 98.21 g m⁻² d⁻¹ for indoor CR unit and 35.47 g m⁻² d⁻¹ for outdoor CR unit. Said et al. (2022) analyzed the relative growth rate of W. globosa using hydroponic fertilizer water. The growth rates in the outdoor experiments ranged from 97.76 to 176.66 g m⁻² d⁻¹ after five days of cultivation. Similarly, Verma and Suthar (2014) treated urban wastewater using Lemna gibba and achieved a maximum relative growth rate of 117.75 g m⁻² d⁻¹. The indoor units showed significantly higher (p < 0.05) growth rates and biomass values from the 7th day compared to the outdoor ones, which can be attributed to the controlled environmental conditions provided to the macrophytes (e.g., regulated temperature and light). Additionally, a biofilm possibly composed of microalgae and bacteria (Cavinatto and Paganini 2007) dispersed through the air, particularly in warm and dry environments, was developed in the outdoor experimental units. Such a phenomenon was not observed in the indoor units, as illustrated in Fig. 6 . The spontaneous formation of biofilms in wastewater is a natural process that occurs when environmental conditions favor the colonization of surfaces by microorganisms, representing a critical mechanism for microbial proliferation (Butler & Boltz, 2014). Factors such as high nutrient concentrations (Rochex and Lebeault 2007; Sehar and Naz 2016; Volk and LeChevallier 1999; Frias et al. 2001), which are often present in effluents, elevated temperature (Adetunji and Odetokun 2012), light intensity (for algae), oxygen availability (Chang et al. 2015), and pH (Agarwal et al. 2011) promote biofilm formation. 3.3 Effluent polishing Starting from the 7th day, the outdoor units exhibited significantly higher values (p < 0.05) for COD and BOD 5,20 parameters, compared to the indoor units. Moreover, the values of these parameters were significantly higher (p < 0.05) in the units used as controls compared to the units containing W. brasiliensis , underscoring their capacity to reduce COD and BOD 5,20 levels (Fig. 7 ). Contrarily to the observations for BOD 5,20 and COD, the external units exhibited significantly lower TN and TP concentrations (p < 0.05) compared to the internal ones (Fig. 8 ). According to Su et al. (2011), biofilms formed by microbial consortia, such as microalgae and bacteria, can achieve a high nutrient reduction efficiency, including TN and TP, reaching up to 93%. Rajasulochana and Preethy (2016) claimed microalgae require nutrients present in wastewater for their growth. The cultivation units with Wolffia showed significantly higher efficiency (p < 0.05) for all parameters analyzed and in both environments, compared to the control units, highlighting the feasibility of using macrophyte as polishing (Fig. 9 ). The highest efficiencies for BOD 5,20 and COD were achieved in the indoor units, whereas the outdoor units showed the best results for TN and TP. The high nutrient removal efficiency in the outdoor units can be attributed to the development of biofilm, which probably hindered macrophyte growth and led to partial plant mortality. The process resulted in the release of decomposing organic matter into the system, contributing to increased concentrations of BOD 5,20 and COD in the outdoor units due to intensified organic degradation. The highest efficiency values for BOD 5,20 and COD were 91% and 76%, respectively, for the internal CW unit. Zanetoni Filho (2019) applied L. punctata for the polishing of domestic effluent and reported maximum removal efficiencies of 66.35% for BOD 5,20 and 59.08% for COD. Kotowska et al. (2018) compared the potential of W. arrhiza and L. minor for treating urban wastewater and reported maximum efficiencies of 91% and 81% for BOD 5,20 removal and 90% and 88% for COD removal, respectively. Garcia (2015) investigated L. punctata for polishing effluents from WWTPs and observed maximum efficiencies of 88.12% for COD and 91.14% for BOD 5,20 . Zhang et al. (2011) applied W. arrhiza in a wastewater tank and observed maximum removal efficiencies of 76.9% for BOD 5,20 and 69.1% for COD. Cerqueira (2021) analyzed L. punctata in polishing aquaculture recirculation system effluent and achieved a maximum COD removal efficiency of 64.5%. The highest phosphorus removal efficiency (TP) observed in this study exceeded 99%. Garcia (2015) reported a maximum TP removal efficiency of 66.18% using L. punctata in WWTP effluent polishing and Valderrama et al. (2002) treated ethanol production wastewater with an integrated culture of Wolffia sp. and Chlorella vulgaris , achieving a maximum TP removal of 28%. Zanetoni Filho (2019) observed a maximum TP removal efficiency of 6.85% and Kotowska et al. (2018) recorded maximum TP removal efficiencies of 83% for W. arrhiza and 77% for L. minor in urban wastewater. Zhang et al. (2011) achieved a maximum TP removal efficiency of 84.3%, whereas Cerqueira (2021) reported 47%. Kumar et al. (2022) analyzed W. globosa cultivated in synthetic wastewater with varying nitrogen concentrations, reporting maximum removal efficiencies of 99.57% for TN and 100% for TP. The highest nitrogen removal efficiency (TN) in the present study exceeded 98%. Xu and Shen (2011) analyzed the cultivation of Spirodela oligorrhiza in swine effluent, achieving a maximum TN removal efficiency of 83.7% and Chen et al. (2018) tested L. punctata in wetland system wastewater and observed an approximately 90% maximum TN removal efficiency. Zanetoni Filho (2019) achieved a maximum TN removal efficiency of 26.76%, whereas Cerqueira (2021) reported 65% for L. punctata . Kotowska et al. (2018) reported maximum TN removal efficiencies of 90% and 78% for W. arrhiza and L. minor , respectively. On the other hand, Zhang et al. (2011) reported a maximum TN removal efficiency of 46.1% with W. arrhiza in a wastewater tank. 4. Conclusions This study assessed the polishing performance of a tropical macrophyte under different environmental conditions for effluent treatment. W. brasiliensis showed high potential for the polishing of effluents from stabilization ponds, particularly under controlled indoor cultivation conditions, with temperature and light regulation favoring macrophyte growth and efficient pollutant removal. The growth rate of W. brasiliensis was strongly influenced by cultivation conditions (particularly nutrient availability in the effluent). In units containing 10% diluted effluent, the growth rate was considerably lower compared to units with undiluted or recirculated effluent, which can be attributed to reduced concentrations of essential nutrients, such as nitrogen and phosphorus, due to dilution. Conversely, in units with undiluted and recirculated effluent, higher nutrient concentrations supported a faster and more efficient growth, resulting in a significantly greater biomass production. The effluent recirculation maintained a continuous and balanced nutrient supply, enhancing both macrophyte growth and pollutant removal efficiency. Biofilm formation in external units can affect macrophyte growth, and this phenomenon underscores a need for strategies that control biofilm growth in macrophyte-based treatment systems towards maximizing the benefits of plant use in effluent polishing. The results obtained in this study indicated that the efficiency in reducing the parameters of COD, BOD 5,20 , TN and TP was similar to that reported in studies conducted in temperate climate regions. However, the observed growth rate was slightly lower, possibly due to differences in local environmental conditions, which may influence the metabolism and adaptation of the organisms involved in the process. Future studies should be developed on a larger scale and under real-world conditions, considering seasonal variability and precipitation influences, for deepening the understanding of W. brasiliensis ' efficiency throughout the year. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. Author Contributions All authors contributed to the study conception and design. Tsunao Matsumoto conceived the ideas for the study; material preparation, data collection and analysis were performed by Ana Beatriz Laluce Vaz. The first draft of the manuscript was written by Ana Beatriz Laluce Vaz, Davi Fernandes Gasparini Cunha and Gleyson Borges Castro and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements: The authors are greateful Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 for supporting this research. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Adetunji VO, Odetokun IA (2012) Biofilm formation in human and tropical foodborne isolates of Listeria strains. American Journal of Food and Technology 7:517-531. https://doi.org/ 10.3923/ajft.2012.517.531 Agarwal RK, Singh S, Bhilegaonkar KN, Singh VP (2011) Optimization of microtitre plate assay for the testing of biofilm formation ability in different Salmonella serotypes. 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Dissertation, Universidade Estadual Paulista Zhang Y, Guifang Y, Feifei L (2011) Purification of water of wastewater pond by Wolffia arrhiza . Journal of Xinyang Normal University (Natural Science Edition) 24:473-475. Ziegler P, Sree KS, Appenroth KJ (2016) Duckweeds for water remediation and toxicity testing. Toxicological & Environmental Chemistry 98:1127-1154. https://doi.org/ 10.1080/02772248.2015.1094701 Ziegler P, Sree KS, Appenroth KJ (2017) The uses of duckweed in relation to water remediation. Desalination and Water Treatment 63:327-342. https://doi.org/ 10.5004/dwt.2017.0479 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 Apr, 2025 Reviewers invited by journal 03 Apr, 2025 Editor invited by journal 02 Mar, 2025 Editor assigned by journal 27 Feb, 2025 First submitted to journal 26 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6099077","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437979387,"identity":"3016b28d-c25f-498e-b8d9-d455aa54d0a2","order_by":0,"name":"Ana Beatriz Laluce Vaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYLCCBwwWQJL5AIidQJyWBAYJIMmWQLIWHgPitPDPbn7AkFAjkdjf3vNN8ucOhjzzBgJaJO4cM2BIOCaROOPM2W3SvGcYimUOELLmRgJQC5tE4gaJ3G3SjG0MiTMI6ZC/kf6BIeEfUIv8m2eSP4nRYnAjx4AhsQ1kCw+bBC8xWgxv5BQcSOyTMJ5xJs3YmveMRLEEIS1yN9I3PvjwzUa2v/3ww5s/d9jkEdQCAgfgLMYGojQgA8YGUnWMglEwCkbBSAAAdJM/cB8XuN0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7248-4433","institution":"Universidade de São Paulo: Universidade de Sao Paulo","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"Beatriz Laluce","lastName":"Vaz","suffix":""},{"id":437979388,"identity":"98cc7d55-64f5-4e52-9b74-e14605c1763e","order_by":1,"name":"Davi Gasparini Fernandes Cunha","email":"","orcid":"","institution":"USP: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Davi","middleName":"Gasparini Fernandes","lastName":"Cunha","suffix":""},{"id":437979389,"identity":"5fc8561e-08a5-46f1-b3e2-70d428187421","order_by":2,"name":"Gleyson B. Castro","email":"","orcid":"","institution":"USP: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Gleyson","middleName":"B.","lastName":"Castro","suffix":""},{"id":437979390,"identity":"ee7c5722-b486-4c91-a538-127fae29de5d","order_by":3,"name":"Tsunao Matsumoto","email":"","orcid":"","institution":"Universidade Estadual Paulista Júlio de Mesquita Filho: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Tsunao","middleName":"","lastName":"Matsumoto","suffix":""}],"badges":[],"createdAt":"2025-02-24 17:50:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6099077/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6099077/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81320151,"identity":"ca495e70-f27f-42b0-a93d-e0f075bad65c","added_by":"auto","created_at":"2025-04-24 17:11:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":603421,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic view of \u003cem\u003eWolffia brasiliensis\u003c/em\u003e at 50x magnification\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/b08404628dceb2d947dd3713.png"},{"id":81319460,"identity":"337bac77-4fb0-442a-96f3-16353b956e0f","added_by":"auto","created_at":"2025-04-24 17:03:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70689,"visible":true,"origin":"","legend":"\u003cp\u003eDetails of the internal cultivation units arranged in the laboratory\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/f7523f54fcfc5b1561618b19.png"},{"id":81320148,"identity":"a638ec9c-5797-45fe-a44f-f2978e6618ea","added_by":"auto","created_at":"2025-04-24 17:11:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11302,"visible":true,"origin":"","legend":"\u003cp\u003eAir temperature values for indoor and outdoor environments\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/728dd3071873489ae7eb6750.png"},{"id":81319456,"identity":"a8a96d07-66b1-43c2-84a7-b9a512d24a6f","added_by":"auto","created_at":"2025-04-24 17:03:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41615,"visible":true,"origin":"","legend":"\u003cp\u003epH values for 35 days of cultivation for indoor and outdoor environments\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/fa88bc04bd9b51d0f94b03e7.png"},{"id":81319458,"identity":"e50caa8e-b3b9-4ddb-860d-98c0b43deb48","added_by":"auto","created_at":"2025-04-24 17:03:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31322,"visible":true,"origin":"","legend":"\u003cp\u003eRelative growth rate and biomass of \u003cem\u003eW. brasiliensis\u003c/em\u003eunder different cultivation conditions (a) results for CW unit (containing only \u003cem\u003eWolffia\u003c/em\u003e) in the indoor environment; (b) results for CW unit (containing only \u003cem\u003eWolffia\u003c/em\u003e) in the outdoor environment; (c) results for ED unit (containing diluted effluent) in the indoor environment; (d) results for ED unit (containing diluted effluent) in the outdoor environment; (e) results for CR unit (containing nutrient recirculation) in the indoor environment; and (f) results for CR unit (containing nutrient recirculation) in the outdoor environment\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/e833867482f2e59f1e00059a.png"},{"id":81320287,"identity":"5018964d-a091-4791-a6e9-8a363e44193d","added_by":"auto","created_at":"2025-04-24 17:19:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":389886,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm formation in (a) outdoor units and \u003cem\u003eWolffia\u003c/em\u003e cultivation in (b) indoor units\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/a80e2b0c89f749dfec874d8c.png"},{"id":81319459,"identity":"acfcb54e-fc86-47f0-a027-a2b310284c09","added_by":"auto","created_at":"2025-04-24 17:03:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":34069,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of COD and BOD\u003csub\u003e5,20\u003c/sub\u003e in the experimental units\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/8f8b38695bba07044a3d28d4.png"},{"id":81320288,"identity":"713b156d-c57b-4870-9acf-aa6c0afccdfb","added_by":"auto","created_at":"2025-04-24 17:19:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":37845,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of Total Nitrogen and Total Phosphorus in the experimental units\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/b46a06a2cc86c88554593b98.png"},{"id":81319468,"identity":"924084ff-1509-4e44-92ea-7324cbcb7997","added_by":"auto","created_at":"2025-04-24 17:03:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":58024,"visible":true,"origin":"","legend":"\u003cp\u003eEfficiency analysis for all experimental units considering BOD\u003csub\u003e5,20\u003c/sub\u003e, COD, TN, and TP\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/3b3314333727192cc3a25d09.png"},{"id":81320999,"identity":"439f5e76-13b5-497b-a2b8-09c40198399a","added_by":"auto","created_at":"2025-04-24 17:27:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2019815,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6099077/v1/aaa640a5-61d0-417b-acb9-46033a699d8d.pdf"}],"financialInterests":"","formattedTitle":"Influence of operational conditions on the efficiency of Wolffia brasiliensis for polishing domestic wastewater of facultative stabilization ponds","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStabilization ponds are among the simplest methods for wastewater treatment because they are characterized by low costs of implementation, maintenance, and operation (Arthur 1983; Von Sperling 2002). Such systems are particularly well-suited for applications in developing countries from tropical and subtropical regions, where land areas are generally available at relatively low prices and climatic conditions such as high temperatures and year-round solar radiation favor the biodegradation process (Arthur 1983; Mahapatra et al. 2022). According to the Brazilian Water Agency (ANA), in 2020, stabilization ponds accounted for 32% of the 3,600 sewage treatment plants (STPs) under operation in Brazil, serving as the primary treatment technology (ANA 2020). However, while such ponds promote a satisfactory removal of organic matter, their efficiency for nutrient removal, mainly regarding nitrogen and phosphorus, is limited (Fujioka et al. 2020), resulting in the eutrophication of receiving water bodies and overall water quality decrease (Miwa et al. 2007). Hence, effluent polishing technologies that offer both high efficiency and low installation costs are often required and have seen increasing adoption. Aquatic macrophytes have emerged as a prominent strategy for nutrient and organic matter abatement from wastewater, being recognized as a versatile and scalable intervention that can be adapted to diverse regional and operational contexts (Herzog and Rozado 2010; Uysal 2013).\u003c/p\u003e \u003cp\u003eAmong aquatic macrophyte species, floating plants of the Lemnaceae family are particularly notable for their simple structural organization and predominance of photosynthetic tissues (Wolverton and McDonald 1980; Ziegler et al. 2016; Chakrabarti et al. 2018). They are tolerant to environmental pollutants, including metals (Cheng and Stomp 2009; Ziegler et al. 2016), and show potential for removing suspended solids and biochemical oxygen demand (Mohedano et al. 2014). Due to such advantageous traits, Lemnaceae species have been increasingly employed in wastewater treatments and post-treatment processes (Ziegler et al. 2017; Toyama et al. 2018). Macrophytes of the genus \u003cem\u003eWolffia\u003c/em\u003e - recognized as the smallest macrophytes in Lemnaceae - are distinguished by their rapid reproduction and growth rates (Suppadit et al. 2008). Kumar et al. (2022) evaluated the nutrient removal capacity of \u003cem\u003eWolffia globosa\u003c/em\u003e under controlled laboratory conditions and reported an above 99% efficiency for removing total inorganic nitrogen and orthophosphate.\u003c/p\u003e \u003cp\u003eSoda et al. (2013) evaluated the nutrient removal capacity of \u003cem\u003eWolffia arrhiza\u003c/em\u003e under controlled laboratory mesocosms supplied with synthetic wastewater and the findings revealed nitrogen and phosphorus removal rates of 0.03\u0026ndash;0.18 mg N m⁻\u0026sup2; d⁻\u0026sup1; and 0.023\u0026ndash;0.079 mg P m⁻\u0026sup2; d⁻\u0026sup1;, respectively. Similarly, Zhang et al. (2011) investigated the potential of \u003cem\u003eW. arrhiza\u003c/em\u003e for polishing effluents from stabilization ponds, reporting removal efficiencies of 84% for total phosphorus (TP), 46% for total nitrogen (TN), 76% for biochemical oxygen demand (BOD₅,20), and 69% for chemical oxygen demand (COD). Numerous studies have underscored the effectiveness of Lemnaceae species in wastewater treatment, particularly in nutrient removal. The most extensively studied genera include \u003cem\u003eLemna\u003c/em\u003e (Valderrama et al. 2002; Uysal 2013; Chen et al. 2018; Toyama et al. 2018), \u003cem\u003eLandoltia\u003c/em\u003e (Garcia 2015; Chen et al. 2018; Toyama et al. 2018; Zanetoni Filho 2019; Cerqueira 2021), and \u003cem\u003eSpirodela\u003c/em\u003e (Cheng and Stomp 2009; Chen et al. 2018; Toyama et al. 2018). In contrast, research on the genus \u003cem\u003eWolffia\u003c/em\u003e remains relatively scarce (Landolt and Kandeler 1987; Kotowska et al. 2018). To date, most investigations on \u003cem\u003eWolffia\u003c/em\u003e have been conducted in continental (Kotowska et al. 2018) and temperate climates (Zhang et al. 2011; Soda et al. 2013; Kumar et al. 2022), with limited data available on its performance under tropical climatic conditions.\u003c/p\u003e \u003cp\u003eThis study evaluated the polishing process of previously treated wastewater from a Wastewater Treatment Plant (WWTP) located in Ilha Solteira (S\u0026atilde;o Paulo State, Brazil) using \u003cem\u003eW. brasiliensis\u003c/em\u003e macrophyte. Three distinct operational conditions, namely, undiluted effluent without recirculation (CW), effluent diluted to 10% without recirculation (ED), and undiluted effluent with recirculation (CR) facilitated by a submersible pump were assessed. The study focused on the influence of operational conditions on the relative growth rate of the macrophyte and the removal efficiency of key contaminants, including chemical oxygen demand (COD), biochemical oxygen demand (BOD\u003csub\u003e5,20\u003c/sub\u003e), total nitrogen (TN), and total phosphorus (TP). The findings offer valuable insights for the design and construction of wetlands, supporting the development of more sustainable solutions in environmental management.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThe study was conducted at the Sanitation Laboratory of the Civil Engineering Department (20\u0026deg;25'42.4\" S 51\u0026deg;20'29.9\" W), at S\u0026atilde;o Paulo State University \u0026ldquo;J\u0026uacute;lio de Mesquita Filho\u0026rdquo; (UNESP), Ilha Solteira campus, S\u0026atilde;o Paulo, Brazil.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Characteristics of the studied wastewater\u003c/h2\u003e \u003cp\u003eThe effluent utilized was collected from the outlet of a primary facultative pond at the Ilha Solteira Wastewater Treatment Plant (WWTP), which includes a preliminary treatment system comprising a screening unit and a sand trap, followed by two primary facultative ponds of 458 m length, 105 m width, and 1.5 m depth (Costa 2015). The initial concentrations of chemical oxygen demand (COD), biochemical oxygen demand (BOD\u003csub\u003e5,20\u003c/sub\u003e), total nitrogen (TN), total phosphorus (TP), turbidity, total solids (TS), total suspended solids (TSS), and dissolved solids (DS) in the effluent were analyzed according to APHA (2017) methods (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInitial characterization of the studied effluent used in the experimental polishing units\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOD (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e371.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBOD\u003csub\u003e5,20\u003c/sub\u003e (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e73.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTN (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e N)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTurbidity (NTU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e220.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTS (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e680.00\u0026thinsp;\u0026plusmn;\u0026thinsp;15.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSS (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e170.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDS (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e510.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote. SD: Standard deviation obtained by three analytical replications.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCOD: Chemical Oxygen Demand; BOD\u003csub\u003e5,20\u003c/sub\u003e: Biochemical Oxygen Demand; TN: Total Nitrogen; TP: Total Phosphorus; TS: Total Solids; TSS: Total Suspended Solids; DS: Dissolved Solids.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant material, microscopic analysis and species identification\u003c/h2\u003e \u003cp\u003eThe macrophyte was collected from the Wastewater Treatment Plant (WWTP) at the Provisional Detention Center of S\u0026atilde;o Jos\u0026eacute; do Rio Preto (20\u0026deg;41'17\" S 49\u0026deg;20'17\" W), S\u0026atilde;o Paulo, Brazil. The WWTP comprises a primary anaerobic pond, a secondary facultative pond, and two maturation ponds in series. \u003cem\u003eWolffia\u003c/em\u003e was found to develop spontaneously at all stages of the treatment process; however, the specimens used in this study were collected from the secondary facultative pond. Following collection, the macrophyte was cultivated and maintained in a 200 L polyethylene tank in an outdoor area adjacent to the laboratory. The tank received natural light and was supplied with the domestic wastewater used in the experimental trials.\u003c/p\u003e \u003cp\u003eAn analysis under a binocular optical microscope (50x magnification) confirmed the macrophyte species selected, whose morphological characteristics, including presence of blackened cells and occurrence of papillae on the upper surface of the plant, were observed. According to Landolt (1994), \u003cem\u003eW. brasiliensis\u003c/em\u003e is distinguished from other species by the presence of darkened cells and fronds of 0.3 to 1.4 mm length. Such features were identified in the specimens used in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Experimental design\u003c/h2\u003e \u003cp\u003eThe assessment of the growth potential of the macrophyte under varying conditions involved analyses of three distinct cultivation scenarios, namely, undiluted effluent without recirculation (CW), effluent diluted to 10% without recirculation (ED), and undiluted effluent with recirculation facilitated by a 2.5 W submersible pump operating at 6 \u0026times; 10⁻\u0026sup3; L s⁻\u0026sup1; continuous flow rate (CR). Additionally, control units containing only effluent (C) were included for evaluations of the self-purification capacity of the effluent and the natural decay of the studied parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The experiment was conducted in two distinct environments. In an indoor one, 12 experimental polyethylene units containing 3 L of effluent each were arranged randomly within a wooden box (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The setup was illuminated by two tubular LED lamps (3000 K, 18 W, and 1.20 m length) connected to an analog photoperiod timer set to a 12 h light: 12 h dark cycle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second environment was an outdoor area adjacent to the laboratory, where 12 experimental units were randomly arranged within a greenhouse of 3 m width, 3 m length, and 2.78 m height. The initial density of fresh \u003cem\u003eWolffia\u003c/em\u003e biomass in all experimental units was standardized at 200 g m⁻\u0026sup2;. The volume of evaporated effluent in each unit was replenished weekly towards maintaining the experimental conditions. The evaporated volume was systematically monitored and recorded, with corrections applied for accurate calculations of the removal efficiencies of the evaluated parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Growth rate\u003c/h2\u003e \u003cp\u003e \u003cem\u003eWolffia\u003c/em\u003e growth was quantified in each experimental unit with the use of Relative Growth Rate (RGR) (g m⁻\u0026sup2; d⁻\u0026sup1;) based on dry matter, as described by Verma and Suthar (2014), and calculated by Eq.\u0026nbsp;1.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:RGR=\\frac{DM}{N.A}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\left[1\\right]$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere DM\u0026thinsp;=\u0026thinsp;dry matter during the growing season (g), N\u0026thinsp;=\u0026thinsp;number of growing days, and A\u0026thinsp;=\u0026thinsp;surface area of ​​the growing section (m\u0026sup2;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Monitoring parameters and statistical analysis\u003c/h2\u003e \u003cp\u003eThe water quality parameters used for evaluations of the efficiency of the effluent polishing process, namely biochemical oxygen demand (BOD\u003csub\u003e5,20\u003c/sub\u003e), total phosphorus (TP), total nitrogen (TN), and chemical oxygen demand (COD), were analyzed weekly over a 35-day period, according to the Standard Methods for the Examination of Water and Wastewater (APHA 2017). The ambient temperature was monitored daily by two digital thermohygrometers that recorded maximum and minimum values, while pH was measured weekly by a portable digital pHmeter from Hanna Instruments, following the guidelines provided by APHA (2017). The determination of the reduction efficiencies of BOD\u003csub\u003e5,20\u003c/sub\u003e, COD, TP, and TN was based on the quantification limits specified by APHA (2017). A data analysis was conducted using a completely randomized design, with factor A representing type of treatment, factor B representing the environment (internal or external), and time as a subdivided plot. A statistical analysis was performed with the use of Sisvar software, applying analysis of variance (ANOVA) and Scott-Knott mean clustering test, both with a 5% significance level. The approach enables assessing the significance of the environment (internal versus external) and the growing conditions within each environment (diluted effluent, undiluted effluent, and effluent with recirculation). All the graphs used in this work were generated using Origin\u0026reg; 2023 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Monitoring parameters\u003c/h2\u003e\n \u003cp\u003eThe external temperature was significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the internal one, with averages of 24\u0026deg;C indoor and 31\u0026deg;C outdoor (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). A greater standard deviation was observed in external temperature, which can be attributed to the natural variability of outdoor environments, The optimal temperature range for \u003cem\u003eWolffia\u003c/em\u003e cultivation is between 17\u0026deg;C-30\u0026deg;C and 17.5\u0026deg;C-34\u0026deg;C according to Skillicorn et al. (1993) and Hasan and Chakrabarti (2009), respectively. The indoor environment showed higher growth rates for \u003cem\u003eW. brasiliensis\u003c/em\u003e compared to the outdoor one, which frequently exceeded the ideal temperature range outlined by Skillicorn et al. (1993) and Hasan and Chakrabarti (2009). Kumar et al. (2022) and Chakrabarti et al. (2018) successfully cultivated \u003cem\u003eW. globosa\u003c/em\u003e under controlled laboratory conditions at 31\u0026deg;C using synthetic effluent and Lemna minor under outdoor conditions at 25\u0026ndash;28\u0026deg;C, respectively, both similar to the conditions observed in this study.\u003c/p\u003e\n \u003cp\u003eOverall, regarding the pH parameter, no significant differences were detected among operation conditions within the same environment on the 7th, 21st, and 28th days (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). However, experimental units in outdoor environments showed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher pH values (9.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3) compared to indoor ones (7.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4) from the 7th day of the experiment (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), highlighting the influence of environmental factors such as temperature and light on pH levels. Skillicorn et al. (1993) defined the optimal pH range for \u003cem\u003eWolffia\u003c/em\u003e development as 7.00\u0026ndash;8.00, while Hasan and Chakrabarti (2009) reported that \u003cem\u003eLemnaceae\u003c/em\u003e species can tolerate pH levels between 5 and 9. Thus, the pH values recorded under indoor laboratory conditions were considered viable and associated with higher macrophyte growth rates. In contrast, lower growth rates coincided with high outdoor temperatures and elevated pH levels. The indoor pH values in this study align with those reported by Kotowska et al. (2018) for \u003cem\u003eW. arrhiza\u003c/em\u003e (7.60\u0026ndash;7.90), which supported satisfactory growth, and with those found by Sirirustananun and Jongput (2021) (7.50\u0026ndash;7.79) for the same species. Kumar et al. (2022) observed optimal pH values when cultivating \u003cem\u003eW. globosa\u003c/em\u003e but reported an increase to 10.3 by the end of the culture, a trend consistent with the present study\u0026apos;s findings for outdoor experimental units.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Relative growth rate\u003c/h2\u003e\n \u003cp\u003eIn both environments, CR showed significantly higher biomass and growth rate values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to CW and ED (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), which can be attributed to the recirculation of nutrients within the system. Conversely, ED showed significantly lower values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to CW and CR, due to reduced nutrient concentrations caused by the dilution process. The maximum relative growth rates for all experimental units were recorded on the 7th day of cultivation, i.e., 85.0 g m⁻\u0026sup2; d⁻\u0026sup1; for indoor CW unit, 32.38 g m⁻\u0026sup2; d⁻\u0026sup1; for outdoor CW unit, 74.04 g m⁻\u0026sup2; d⁻\u0026sup1; for indoor ED unit, and 26.19 g m⁻\u0026sup2; d⁻\u0026sup1; for outdoor ED unit, as well as 98.21 g m⁻\u0026sup2; d⁻\u0026sup1; for indoor CR unit and 35.47 g m⁻\u0026sup2; d⁻\u0026sup1; for outdoor CR unit.\u003c/p\u003e\n \u003cp\u003eSaid et al. (2022) analyzed the relative growth rate of \u003cem\u003eW. globosa\u003c/em\u003e using hydroponic fertilizer water. The growth rates in the outdoor experiments ranged from 97.76 to 176.66 g m⁻\u0026sup2; d⁻\u0026sup1; after five days of cultivation. Similarly, Verma and Suthar (2014) treated urban wastewater using \u003cem\u003eLemna gibba\u003c/em\u003e and achieved a maximum relative growth rate of 117.75 g m⁻\u0026sup2; d⁻\u0026sup1;.\u003c/p\u003e\n \u003cp\u003eThe indoor units showed significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) growth rates and biomass values from the 7th day compared to the outdoor ones, which can be attributed to the controlled environmental conditions provided to the macrophytes (e.g., regulated temperature and light). Additionally, a biofilm possibly composed of microalgae and bacteria (Cavinatto and Paganini 2007) dispersed through the air, particularly in warm and dry environments, was developed in the outdoor experimental units. Such a phenomenon was not observed in the indoor units, as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. The spontaneous formation of biofilms in wastewater is a natural process that occurs when environmental conditions favor the colonization of surfaces by microorganisms, representing a critical mechanism for microbial proliferation (Butler \u0026amp; Boltz, 2014). Factors such as high nutrient concentrations (Rochex and Lebeault 2007; Sehar and Naz 2016; Volk and LeChevallier 1999; Frias et al. 2001), which are often present in effluents, elevated temperature (Adetunji and Odetokun 2012), light intensity (for algae), oxygen availability (Chang et al. 2015), and pH (Agarwal et al. 2011) promote biofilm formation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Effluent polishing\u003c/h2\u003e\n \u003cp\u003eStarting from the 7th day, the outdoor units exhibited significantly higher values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for COD and BOD\u003csub\u003e5,20\u003c/sub\u003e parameters, compared to the indoor units. Moreover, the values of these parameters were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the units used as controls compared to the units containing \u003cem\u003eW. brasiliensis\u003c/em\u003e, underscoring their capacity to reduce COD and BOD\u003csub\u003e5,20\u003c/sub\u003e levels (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eContrarily to the observations for BOD\u003csub\u003e5,20\u003c/sub\u003e and COD, the external units exhibited significantly lower TN and TP concentrations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the internal ones (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). According to Su et al. (2011), biofilms formed by microbial consortia, such as microalgae and bacteria, can achieve a high nutrient reduction efficiency, including TN and TP, reaching up to 93%. Rajasulochana and Preethy (2016) claimed microalgae require nutrients present in wastewater for their growth.\u003c/p\u003e\n \u003cp\u003eThe cultivation units with \u003cem\u003eWolffia\u003c/em\u003e showed significantly higher efficiency (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for all parameters analyzed and in both environments, compared to the control units, highlighting the feasibility of using macrophyte as polishing (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The highest efficiencies for BOD\u003csub\u003e5,20\u003c/sub\u003e and COD were achieved in the indoor units, whereas the outdoor units showed the best results for TN and TP. The high nutrient removal efficiency in the outdoor units can be attributed to the development of biofilm, which probably hindered macrophyte growth and led to partial plant mortality. The process resulted in the release of decomposing organic matter into the system, contributing to increased concentrations of BOD\u003csub\u003e5,20\u003c/sub\u003e and COD in the outdoor units due to intensified organic degradation.\u003c/p\u003e\n \u003cp\u003eThe highest efficiency values for BOD\u003csub\u003e5,20\u003c/sub\u003e and COD were 91% and 76%, respectively, for the internal CW unit. Zanetoni Filho (2019) applied \u003cem\u003eL. punctata\u003c/em\u003e for the polishing of domestic effluent and reported maximum removal efficiencies of 66.35% for BOD\u003csub\u003e5,20\u003c/sub\u003e and 59.08% for COD. Kotowska et al. (2018) compared the potential of \u003cem\u003eW. arrhiza\u003c/em\u003e and \u003cem\u003eL. minor\u003c/em\u003e for treating urban wastewater and reported maximum efficiencies of 91% and 81% for BOD\u003csub\u003e5,20\u003c/sub\u003e removal and 90% and 88% for COD removal, respectively. Garcia (2015) investigated \u003cem\u003eL. punctata\u003c/em\u003e for polishing effluents from WWTPs and observed maximum efficiencies of 88.12% for COD and 91.14% for BOD\u003csub\u003e5,20\u003c/sub\u003e. Zhang et al. (2011) applied \u003cem\u003eW. arrhiza\u003c/em\u003e in a wastewater tank and observed maximum removal efficiencies of 76.9% for BOD\u003csub\u003e5,20\u003c/sub\u003e and 69.1% for COD. Cerqueira (2021) analyzed \u003cem\u003eL. punctata\u003c/em\u003e in polishing aquaculture recirculation system effluent and achieved a maximum COD removal efficiency of 64.5%.\u003c/p\u003e\n \u003cp\u003eThe highest phosphorus removal efficiency (TP) observed in this study exceeded 99%. Garcia (2015) reported a maximum TP removal efficiency of 66.18% using \u003cem\u003eL. punctata\u003c/em\u003e in WWTP effluent polishing and Valderrama et al. (2002) treated ethanol production wastewater with an integrated culture of \u003cem\u003eWolffia sp.\u003c/em\u003e and \u003cem\u003eChlorella vulgaris\u003c/em\u003e, achieving a maximum TP removal of 28%. Zanetoni Filho (2019) observed a maximum TP removal efficiency of 6.85% and Kotowska et al. (2018) recorded maximum TP removal efficiencies of 83% for \u003cem\u003eW. arrhiza\u003c/em\u003e and 77% for \u003cem\u003eL. minor\u003c/em\u003e in urban wastewater. Zhang et al. (2011) achieved a maximum TP removal efficiency of 84.3%, whereas Cerqueira (2021) reported 47%. Kumar et al. (2022) analyzed \u003cem\u003eW. globosa\u003c/em\u003e cultivated in synthetic wastewater with varying nitrogen concentrations, reporting maximum removal efficiencies of 99.57% for TN and 100% for TP.\u003c/p\u003e\n \u003cp\u003eThe highest nitrogen removal efficiency (TN) in the present study exceeded 98%. Xu and Shen (2011) analyzed the cultivation of \u003cem\u003eSpirodela oligorrhiza\u003c/em\u003e in swine effluent, achieving a maximum TN removal efficiency of 83.7% and Chen et al. (2018) tested \u003cem\u003eL. punctata\u003c/em\u003e in wetland system wastewater and observed an approximately 90% maximum TN removal efficiency. Zanetoni Filho (2019) achieved a maximum TN removal efficiency of 26.76%, whereas Cerqueira (2021) reported 65% for \u003cem\u003eL. punctata\u003c/em\u003e. Kotowska et al. (2018) reported maximum TN removal efficiencies of 90% and 78% for \u003cem\u003eW. arrhiza\u003c/em\u003e and \u003cem\u003eL. minor\u003c/em\u003e, respectively. On the other hand, Zhang et al. (2011) reported a maximum TN removal efficiency of 46.1% with \u003cem\u003eW. arrhiza\u003c/em\u003e in a wastewater tank.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study assessed the polishing performance of a tropical macrophyte under different environmental conditions for effluent treatment. \u003cem\u003eW. brasiliensis\u003c/em\u003e showed high potential for the polishing of effluents from stabilization ponds, particularly under controlled indoor cultivation conditions, with temperature and light regulation favoring macrophyte growth and efficient pollutant removal. The growth rate of \u003cem\u003eW. brasiliensis\u003c/em\u003e was strongly influenced by cultivation conditions (particularly nutrient availability in the effluent). In units containing 10% diluted effluent, the growth rate was considerably lower compared to units with undiluted or recirculated effluent, which can be attributed to reduced concentrations of essential nutrients, such as nitrogen and phosphorus, due to dilution. Conversely, in units with undiluted and recirculated effluent, higher nutrient concentrations supported a faster and more efficient growth, resulting in a significantly greater biomass production. The effluent recirculation maintained a continuous and balanced nutrient supply, enhancing both macrophyte growth and pollutant removal efficiency. Biofilm formation in external units can affect macrophyte growth, and this phenomenon underscores a need for strategies that control biofilm growth in macrophyte-based treatment systems towards maximizing the benefits of plant use in effluent polishing. The results obtained in this study indicated that the efficiency in reducing the parameters of COD, BOD\u003csub\u003e5,20\u003c/sub\u003e, TN and TP was similar to that reported in studies conducted in temperate climate regions. However, the observed growth rate was slightly lower, possibly due to differences in local environmental conditions, which may influence the metabolism and adaptation of the organisms involved in the process. Future studies should be developed on a larger scale and under real-world conditions, considering seasonal variability and precipitation influences, for deepening the understanding of \u003cem\u003eW. brasiliensis\u003c/em\u003e' efficiency throughout the year.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES) \u0026ndash; Finance Code 001.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Tsunao Matsumoto conceived the ideas for the study; material preparation, data collection and analysis were performed by Ana Beatriz Laluce Vaz. The first draft of the manuscript was written by Ana Beatriz Laluce Vaz, Davi Fernandes Gasparini Cunha and Gleyson Borges Castro and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThe authors are greateful Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES) \u0026ndash; Finance Code 001 for supporting this research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdetunji VO, Odetokun IA (2012) Biofilm formation in human and tropical foodborne isolates of Listeria strains. 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Desalination and Water Treatment 63:327-342. https://doi.org/ 10.5004/dwt.2017.0479\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"wetlands","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wela","sideBox":"Learn more about [Wetlands](https://www.springer.com/journal/13157)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/wela/default.aspx","title":"Wetlands","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Wastewater treatment, Organic matter removal, Nutrient removal, Lemnaceae, Macrophyte","lastPublishedDoi":"10.21203/rs.3.rs-6099077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6099077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStabilization ponds are widely used for wastewater treatment in developing countries but have limitations in removing certain pollutants, necessitating polishing processes to enhance effluent quality and mitigate environmental impacts. Aquatic macrophytes, particularly \u003cem\u003eLemnaceae\u003c/em\u003e, offer a low-cost, efficient alternative due to their rapid reproduction and high nutrient absorption. This study evaluated the effects of operational conditions on \u003cem\u003eWolffia brasiliensis\u003c/em\u003e development and its efficiency in polishing wastewater from facultative stabilization ponds. The parameters assessed were chemical oxygen demand (COD), biochemical oxygen demand (BOD₅,₂₀), total nitrogen (TN), and total phosphorus (TP). Three cultivation conditions were tested: untreated effluent, recirculated effluent (6 \u0026times; 10⁻\u0026sup3; L s⁻\u0026sup1; flow rate), and 10% diluted effluent. Experiments were conducted indoors (21,818 lux, 24\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u0026deg;C) and outdoors (natural conditions, 31\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u0026deg;C). Biofilm formation hindered macrophyte growth and contaminant removal in outdoor units. Indoor conditions yielded higher biomass (85.87\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9 g m⁻\u0026sup2; d⁻\u0026sup1;) than outdoor ones (31.35\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 g m⁻\u0026sup2; d⁻\u0026sup1;). Recirculated effluent led to the highest growth rates (42.06\u0026thinsp;\u0026plusmn;\u0026thinsp;33.1 g m⁻\u0026sup2; d⁻\u0026sup1; indoor, 15.70\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7 g m⁻\u0026sup2; d⁻\u0026sup1; outdoor). All \u003cem\u003eW. brasiliensis\u003c/em\u003e units significantly improved pollutant removal compared to controls. These findings highlight \u003cem\u003eW. brasiliensis\u003c/em\u003e as an effective, sustainable solution for polishing effluents from facultative stabilization ponds, particularly for BOD₅,₂₀, COD, TN, and TP removal, reinforcing its potential for wastewater management in developing countries.\u003c/p\u003e","manuscriptTitle":"Influence of operational conditions on the efficiency of Wolffia brasiliensis for polishing domestic wastewater of facultative stabilization ponds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 17:03:32","doi":"10.21203/rs.3.rs-6099077/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-04T05:11:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T13:27:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Wetlands","date":"2025-03-02T18:55:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-28T04:04:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Wetlands","date":"2025-02-27T04:44:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"wetlands","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wela","sideBox":"Learn more about [Wetlands](https://www.springer.com/journal/13157)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/wela/default.aspx","title":"Wetlands","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2790f4a9-c6f1-4ca8-b1c1-3d78bc27c9be","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T19:53:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-24 17:03:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6099077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6099077","identity":"rs-6099077","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}