Mixed-species aquatic weed fertilizers enhance Brassica rapa growth through nutrient synergy within circular nutrient recovery systems

Mixed-species aquatic weed fertilizers enhance Brassica rapa growth through nutrient synergy within circular nutrient recovery systems | 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 Article Mixed-species aquatic weed fertilizers enhance Brassica rapa growth through nutrient synergy within circular nutrient recovery systems Cedric Mankponse Antoine Assogba, Nakashima Yoshitaka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8796299/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Submerged aquatic weeds pose persistent management challenges in freshwater ecosystems, yet their rapid biomass accumulation and nutrient-rich composition represent an underutilized resource for sustainable agriculture. Valorizing this biomass may help link freshwater ecosystem management with circular nutrient recovery and climate-resilient food systems. This study evaluated the agronomic potential of three dominant submerged macrophytes Egeria densa , Elodea nuttallii , and Vallisneria sp as organic fertilizers for Brassica rapa L. var. perviridis , with particular emphasis on comparing single-species and mixed-species formulations integrated with locally available organic residues. Results: A greenhouse experiment was conducted using a completely randomized design with 26 treatments, including four aquatic-weed–based fertilizers (three single-species and one mixed-species), applied at three dosage levels and in two application forms (raw solid and liquid extract), alongside unfertilized and standard commercial controls. Fertilizer identity emerged as the primary determinant of plant performance, consistently outweighing the effects of application dose and form. Across biomass production and morphological traits, the mixed-species fertilizer produced the highest and most stable responses, particularly in shoot fresh and dry biomass, plant height, and leaf production. Single-species fertilizers showed intermediate and species-dependent performance, while Vallisneria -based formulations consistently resulted in lower growth. Dose effects were secondary and fertilizer-dependent, and application form had minimal influence. Growth enhancement was primarily driven by increased aboveground biomass and canopy development, while root biomass, shoot–root allocation, and leaf chlorophyll index remained largely unchanged. Conclusions: These results demonstrate that combining multiple submerged aquatic weed species into a single fertilizer formulation confers clear agronomic advantages over single-species applications. Mixed-species aquatic weed fertilizers enhance nutrient-use efficiency and crop growth without increasing application complexity, supporting their potential role in circular nutrient recovery strategies, sustainable vegetable production, and climate-resilient agricultural systems under increasing environmental variability. Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Plant sciences aquatic weeds Egeria densa Elodea nuttalli Vallisneria sp. waste valorization organic fertilizer circular bioeconomy climate change resource recycling Brassica rapa L. var. perviridis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Global food production systems are increasingly constrained by climate change, nutrient pollution, and accelerating degradation of terrestrial and freshwater ecosystems. These pressures compromise nutrient cycling efficiency, freshwater ecosystem functioning, and long-term agricultural productivity, while intensifying trade-offs between food security and environmental protection (Rockström et al. 2009 ; FAO 2017; IPCC 2023 ). Addressing these interconnected challenges requires nutrient management strategies that simultaneously support crop production, reduce environmental burdens, and enhance resilience under climate variability, directly contributing to multiple Sustainable Development Goals (United Nations 2015 ). Submerged aquatic weeds (macrophytes) represent a persistent environmental management challenge in freshwater ecosystems worldwide, particularly under conditions of nutrient enrichment and altered hydrology. In Japan and many temperate regions, species such as Egeria densa , Elodea nuttallii , and Vallisneria sp. proliferate rapidly in eutrophic waters, forming dense stands that obstruct waterways, disrupt hydrological processes, reduce dissolved oxygen availability, and displace native aquatic biodiversity. Management typically relies on mechanical harvesting, which is costly, recurrent, and often addresses symptoms rather than underlying nutrient drivers of weed proliferation (Asaeda et al. 2020 ). As a result, large quantities of harvested aquatic biomass are commonly treated as waste despite their substantial accumulation of nutrients and organic matter (Zoppi et al. 2024 ). From a circular nutrient management perspective, submerged aquatic weed biomass represents a largely untapped resource. Aquatic macrophytes actively assimilate nitrogen, phosphorus, potassium, and micronutrients from water and sediments, functioning as effective nutrient sinks in freshwater systems (Muñoz Escobar et al. 2011 ; Zoppi et al. 2024 ). Their removal therefore intersects directly with eutrophication mitigation and nutrient recovery, processes expected to become increasingly important under climate change scenarios characterized by intensified nutrient loading and hydrological variability (Dissanayaka et al. 2023 ). In addition, organic fertilizer systems frequently incorporate co-amendments derived from agricultural by-products, food processing residues, and mineral sources to stimulate microbial activity, enhance nutrient retention, and stabilize soil chemical conditions (Lizundia et al. 2022 ). Cereal by-products such as rice bran and food residues such as spent coffee grounds have been shown to improve nutrient availability and crop performance through their effects on microbial processes and gradual nutrient release, while calcium-rich materials such as oyster shell powder contribute to pH buffering and nutrient balance (Kang et al. 2008 ; Alauddin et al. 2020 ; Birnbaum et al. 2025 ; Li et al. 2025 ; Zhang et al. 2025 ; Kumar et al. 2025 ). Integrating submerged aquatic weed biomass within such composite organic matrices reflects realistic fertilization practices and allows the functional role of aquatic biomass to be evaluated within operationally relevant nutrient systems. In parallel, although aquatic weed biomass has attracted increasing attention as a potential organic fertilizer or compost feedstock, existing studies have largely focused on single-species applications or nutrient characterization, with limited emphasis on mixed-species biomass applications or composts. This contrasts with real-world harvesting conditions, where aquatic weed biomass typically consists of mixed-species assemblages rather than monospecific stands. Evidence from litter decomposition and organic amendment research demonstrates that mixing chemically and structurally diverse plant residues can generate non-additive, synergistic effects on decomposition rates, nutrient mineralization, and plant-available nutrient pools. These effects arise from complementary nutrient profiles, heterogeneous substrates, and enhanced microbial interactions, resulting in more balanced and temporally buffered nutrient release (Gartner and Cardon 2004 ; Hättenschwiler et al. 2005 ; Porre et al. 2020 ). Despite this well-established ecological foundation, the agronomic implications of mixed-species aquatic weed fertilizers, particularly in short-cycle vegetable systems, remain poorly understood. Moreover, the relative importance of fertilizer composition relative to application dose and preparation form has not been systematically resolved. Addressing these knowledge gaps is particularly relevant in the context of climate-responsive and circular-economy-oriented nutrient management strategies, where resilience, efficiency, and adaptability of organic inputs are increasingly valued (Khanna et al. 2024 ). Mixed-species organic fertilizers may offer greater buffering capacity against variability in biomass availability, nutrient composition, and environmental conditions, thereby enhancing the robustness of nutrient recycling systems. If effective, such approaches could simultaneously support freshwater ecosystem management, reduce organic waste disposal pressures, improve nutrient-use efficiency, and contribute to sustainable crop production under increasing climatic uncertainty. Against this background, the present study evaluated the agronomic performance of fertilizers derived from submerged aquatic weeds Egeria densa , Elodea nuttallii , and Vallisneria sp. , applied as single-species or mixed-species formulations and combined with locally available organic co-amendments. Using Brassica rapa L. var. perviridis as a model short-cycle leafy vegetable, this study assessed the effects of fertilizer type, application dose, and application form on plant biomass production, morphological development, biomass allocation, and physiological status under controlled greenhouse conditions. By linking aquatic weed valorization with crop productivity, this study provides experimental evidence supporting circular nutrient recovery strategies at the interface of freshwater ecosystem management, sustainable agriculture, and climate-responsive environmental stewardship. 2. Materials and methods 2.1 Aquatic weed biomass sources and nutrient profiles Biomass of three submerged macrophyte species commonly occurring in Japanese freshwater ecosystems, Egeria densa , Elodea nuttallii , and Vallisneria sp ., was collected from waterways in Urayasu, Okayama Prefecture, Japan, in January 2025 (Table S0.1). Collected biomass was rinsed with tap water to remove adhering sediments and debris, oven-dried at 60°C to constant weight, and ground using a Wonder Blender WB-1 from Osaka Chemical Co. Ltd. prior to fertilizer preparation. Baseline nutrient composition data for each macrophyte species and organic residues, including nitrogen (N), phosphorus (P), and potassium (K) were obtained from published literature. These data were used to characterize relative nutrient profiles among species and to inform fertilizer formulation and component ratios. A summary of the nutrient characteristics of each species and organic residues is provided in Table 1 . Total N, P, and K are expressed on an average percentage dry weight basis. Table 1 Nutrient composition of submerged macrophyte species and organic inputs used for fertilizer formulation. N (%) P (%) K (%) Source Egeria densa 3.28 0.60 3.84 (Oki and Une, 1989 ) Elodea nuttalli 3.90 0.49 3.75 (Oki and Une, 1989 ) Vallisneria natans 2.99 0.48 3.87 (Oki and Une, 1989 ) Rice husk 0.47 0.05 25.98 (Abbas et al. 2012 ) Spent coffee grounds 2.20 0.10 0.50 (Birnbaum et al. 2025 ) 2.2 Fertilizer formulation and preparation All fertilizer treatments were prepared using a standardized organic base mixture to ensure comparability across treatments. The master dry fertilizer mix consisted of 65% aquatic weed biomass, 15% rice bran, 15% spent coffee grounds, and 5% oyster shell powder (w/w). The aquatic biomass fraction constituted the primary nutrient source for the different fertilizer treatments and represented the experimental factor of interest. Rice bran and spent coffee grounds were included as readily available organic co-substrates to enhance microbial activity and nutrient mineralization, while oyster shell powder was incorporated at a low proportion as a calcium source and pH-buffering agent. The ratio was selected to balance nutrient supply, decomposition rate, and material stability while avoiding excessive alkalinity or nutrient immobilization. For single-species fertilizers, the aquatic biomass fraction comprised exclusively one macrophyte species ( E. densa , E. nuttallii , or Vallisneria sp. ). For the mixed-species fertilizer, the biomass fraction was composed of equal proportions of the three species. Rice bran and coffee grounds were air-dried prior to mixing, and all components were thoroughly homogenized before application. 2.3 Experimental design and treatment structure The experiment followed a completely randomized design (CRD) with four replicates per treatment, yielding a total of 104 experimental units (26 treatments × 4 replicates), each consisting of one plant. Twenty-four treatments were arranged in a 4 × 3 × 2 factorial structure combining four fertilizer types, three application doses, and two application forms. Fertilizer types were coded as ED ( Egeria densa ), EN ( Elodea nuttallii ), VA ( Vallisneria sp. ), and MX ( mixed species ); dose levels as low (L), optimum (O), and high (H); and application forms as raw solid (R) or liquid extract (E). Two additional controls were included: an unfertilized control (CTRL) and a standard commercial control (COMM) consisting of a pre-fertilized seed-sowing soil commonly used for leafy vegetable cultivation in Okayama. Treatment allocation was fully randomized at the start of the experiment, and the randomized arrangement was maintained after transfer from the incubator to the greenhouse. Treatment codes and descriptions are summarized in Table 2 . Table 2 List of experiment treatments No. Treatment label Fertilizer identity Dose level Application form Description 1 CTRL – – – Unfertilized control 2 COMM – – – Standard commercial control 3 ED-L-R Egeria densa Low Raw Egeria-based fertilizer, low dose, raw solid 4 ED-O-R Egeria densa Optimum Raw Egeria-based fertilizer, optimum dose, raw solid 5 ED-H-R Egeria densa High Raw Egeria-based fertilizer, high dose, raw solid 6 ED-L-E Egeria densa Low Extract Egeria-based fertilizer, low dose, liquid extract 7 ED-O-E Egeria densa Optimum Extract Egeria-based fertilizer, optimum dose, liquid extract 8 ED-H-E Egeria densa High Extract Egeria-based fertilizer, high dose, liquid extract 9 EN-L-R Elodea nuttallii Low Raw Elodea-based fertilizer, low dose, raw solid 10 EN-O-R Elodea nuttallii Optimum Raw Elodea-based fertilizer, optimum dose, raw solid 11 EN-H-R Elodea nuttallii High Raw Elodea-based fertilizer, high dose, raw solid 12 EN-L-E Elodea nuttallii Low Extract Elodea-based fertilizer, low dose, liquid extract 13 EN-O-E Elodea nuttallii Optimum Extract Elodea-based fertilizer, optimum dose, liquid extract 14 EN-H-E Elodea nuttallii High Extract Elodea-based fertilizer, high dose, liquid extract 15 VA-L-R Vallisneria sp. Low Raw Vallisneria-based fertilizer, low dose, raw solid 16 VA-O-R Vallisneria sp. Optimum Raw Vallisneria-based fertilizer, optimum dose, raw solid 17 VA-H-R Vallisneria sp. High Raw Vallisneria-based fertilizer, high dose, raw solid 18 VA-L-E Vallisneria sp. Low Extract Vallisneria-based fertilizer, low dose, liquid extract 19 VA-O-E Vallisneria sp. Optimum Extract Vallisneria-based fertilizer, optimum dose, liquid extract 20 VA-H-E Vallisneria sp. High Extract Vallisneria-based fertilizer, high dose, liquid extract 21 MX-L-R Mixed species Low Raw Mixed-species fertilizer, low dose, raw solid 22 MX-O-R Mixed species Optimum Raw Mixed-species fertilizer, optimum dose, raw solid 23 MX-H-R Mixed species High Raw Mixed-species fertilizer, high dose, raw solid 24 MX-L-E Mixed species Low Extract Mixed-species fertilizer, low dose, liquid extract 25 MX-O-E Mixed species Optimum Extract Mixed-species fertilizer, optimum dose, liquid extract 26 MX-H-E Mixed species High Extract Mixed-species fertilizer, high dose, liquid extract 2.4 Soil substrate and pot preparation Field soil was collected from the Okayama University Field Research Center, air-dried, and sieved to remove coarse debris. During the incubator phase, each pot was filled with 300 g of air-dried soil and fitted with drainage holes to prevent waterlogging. After transplanting to larger pots for the greenhouse phase, each pot was filled with approximately 1.52 kg of the same soil to provide increased root volume while maintaining consistency in soil properties across growth stages. 2.5 Fertilizer dosage levels and application forms Each fertilizer type was applied at three dosage levels based on soil dry weight: low (0.5%, 1.5 g per pot), optimum (1.0%, 3.0 g per pot), and high (2.0%, 6.0 g per pot). Fertilizers were applied either as raw solid material or as a liquid extract prepared to deliver an equivalent total fertilizer mass per pot. Liquid fertilizer extracts were prepared by mixing the required fertilizer mass with distilled water at a 1:10 (w/v) ratio, heating at 95°C for 30 min, cooling to room temperature, and filtering to remove solids. Fresh extracts were prepared prior to each application to minimize microbial alteration of nutrient composition. 2.6 Plant material and growth conditions Seeds of B. rapa var. perviridis were surface-sterilized and germinated under controlled conditions using a petri dish. Uniform seedlings were transplanted 6 days after gemination at the one-true-leaf stage into prepared pots. For the first 35 days, plants were grown in an incubator to ensure uniform early establishment, using V-shaped plastic nursery pots (height 7 cm, bottom diameter 6 cm, top diameter 9 cm). Incubator conditions were maintained at 25°C during the light period and 20°C during the dark period under a 12 h light: 12 h dark photoperiod. Raw fertilizer treatments were applied once as a top-dressing 7 days after germination. In contrast, liquid fertilizer extract treatments were applied twice weekly (every 3 days) for approximately four weeks, starting on day 7 after germination, resulting in seven applications in total. For extract treatments, the assigned fertilizer dose was divided evenly among applications. From day 36, all treatments per pot were transplanted into larger V-shaped plastic nursery pots (height 12 cm, bottom diameter 10 cm, top diameter 15 cm) and transferred to a greenhouse. Plants were grown under natural light and temperature conditions until harvest on day 63. The total experiment run from 18 February to 22 April 2025. 2.7 Growth measurements Plant height, leaf number, and chlorophyll index (SPAD) were measured weekly throughout the experiment. At harvest, shoot and root fresh weight were recorded, followed by oven-drying at 60°C to constant weight to determine shoot and root dry biomass. 2.8 Statistical analysis Data analysis followed a two-step approach reflecting the experimental structure. First, a three-way factorial analysis of variance (ANOVA) was conducted on the 24 factorial treatments to assess the effects of fertilizer type, application dose, application form, and their interactions. Second, a one-way ANOVA including all 26 treatments was performed to compare fertilizer treatments with the unfertilized and commercial controls. Post-hoc comparisons were performed using Tukey’s honest significant difference (HSD) test, and selected contrasts against control treatments were examined using Dunnett-type comparisons. Model assumptions were evaluated using residual diagnostics, and response variables violating normality were log-transformed prior to analysis. All statistical analyses were conducted using R software (version 4.5.2). 3. Results 3.1 Overall plant responses to aquatic-weed–based fertilizers Across all measured variables, including biomass production, plant height, leaf number, and chlorophyll index (SPAD), clear differences in plant growth were observed among fertilizer treatments (Fig. 1 ). Visual inspection prior to harvest indicated marked variation in aboveground development across fertilizer types, with mixed-species (MX) treatments exhibiting more uniform canopy development than single-species treatments, particularly those based on Vallisneria sp. 3.2 Effects of fertilizer treatments on total plant biomass Total plant biomass responded significantly to fertilizer identity (type). Factorial ANOVA revealed a significant main effect of fertilizer type on both total fresh weight ( p = 0.0037) and total dry weight ( p = 0.019), whereas dose, application form, and interaction terms were not significant (Tables S1.1 and S1.2). Across all treatments, the mixed-species fertilizer (MX) produced the highest total biomass, followed by Elodea nuttallii –based (EN) and Egeria densa –based (ED) fertilizers, while Vallisneria sp –based fertilizer (VA) consistently resulted in the lowest biomass accumulation. When all 26 treatments were compared, significant differences in total fresh and dry biomass were detected (Tables S1.3 and S1.4). Several MX-based treatments and high-performing EN treatments occupied the upper end of the biomass distribution, whereas VA treatments were consistently among the lowest. Control treatments generally fell within an intermediate range, overlapping with multiple fertilizer treatments (Figs. 2 and 3 ). 3.3 Effects of fertilizer treatments on shoot biomass production Shoot biomass production was strongly influenced by fertilizer composition, with consistent patterns observed for both fresh and dry biomass. Factorial ANOVA revealed fertilizer type as a significant main effect for shoot fresh weight ( p = 0.001) and shoot dry weight ( p = 0.0067), along with significant fertilizer type × dose interactions for both variables (fresh weight: p = 0.026; dry weight: p = 0.046, Tables S2.1 and S2.2). Across all application doses, the mixed-species fertilizer (MX) consistently produced the highest shoot biomass, significantly outperforming single-species fertilizers at low and optimum doses (Fig. 4 ). Among single-species fertilizers, Egeria densa –based (ED) and Elodea nuttallii –based (EN) fertilizers exhibited moderate increases in shoot biomass at the highest application rate, whereas the Vallisneria sp.–based fertilizer (VA) showed limited responsiveness to increasing dose. At the highest application level, differences in shoot dry biomass among MX, ED, and EN-treatments were reduced, while VA-treatments consistently produced the lowest shoot biomass. 3.4 Root biomass responses to fertilizer treatments In contrast to aboveground biomass, root biomass showed limited responsiveness to fertilizer treatments. Factorial ANOVA detected no significant effects of fertilizer type, dose, application form, or their interactions on either root fresh or root dry biomass (Tables S3.1 and S3.2). Although one-way ANOVA across all treatments indicated significant overall differences (fresh weight p = 0. 0179 and dry weight p = 0.0167), post hoc comparisons revealed substantial overlap among treatments, including both controls (Tables S3.3 - S3.6). 3.5 Biomass allocation patterns (shoot to root ratios) Shoot-to-root biomass allocation remained largely conserved across fertilizer treatments. After log transformation, factorial ANOVA revealed no significant effects of fertilizer type, dose, application form, or their interactions on either fresh or dry shoot:root ratios (Tables S4.1 and S4.2). When all treatments were compared, significant variation in shoot:root fresh ( p = 0.0003) and dry ( p = 0.0006) weight ratios was detected (Tables S4.3 and S4.4). On a fresh weight basis, several MX treatments exhibited higher shoot allocation relative to the unfertilized control. However, overall allocation patterns remained largely conserved across treatments (Tables S4.5 and S4.6). 3.6 Effects of aquatic-weed fertilizers on plant morphology and physiological status Plant morphological traits responded clearly to fertilizer composition. In the factorial analysis, fertilizer type exerted a significant effect on plant height ( p = 0.0012) and leaf number ( p = 0.0082), whereas application dose and application form showed no consistent main effects. Interaction effects were marginal ( p = 0.056) or non-significant (Tables S5.1 and S5.2). Across fertilizer types, the mixed-species fertilizer (MX) consistently resulted in taller plants and greater leaf production, followed by the Elodea- and Egeria-based fertilizers (EN and ED), while the Vallisneria-based fertilizer (VA) produced the smallest plants. When all 26 treatments were compared, both plant height and leaf number differed significantly among treatments (Tables S5.3 and S5.4). Raw solid MX treatments and selected high-performing EN and liquid-extract MX treatments consistently achieved the highest plant heights, exceeding the unfertilized control (CTRL). For leaf number, low and optimum doses of raw solid MX treatments ranked highest and outperformed the commercial fertilizer control. In contrast, Vallisneria-based (VA) treatments consistently clustered among the lowest values for both traits (Figs. 5 and 6 ). In contrast to morphological traits, leaf chlorophyll index (SPAD) remained comparatively stable across treatments. Fertilizer type, dose, and application form did not significantly affect SPAD values in the factorial analysis (Table S5.5). Although significant differences were detected when all treatments were compared ( p = 0.0148), post-hoc separation was limited, and SPAD values varied within a narrow range (Tables S5.6 and S5.7). 4. Discussion This study demonstrates that aquatic weed–based fertilizers derived from submerged macrophytes ( Egeria densa , Elodea nuttallii , and Vallisneria sp.) can effectively support the growth of Brassica rapa , with fertilizer identity emerging as the dominant determinant of plant performance. Across biomass production, morphological development, and physiological indicators, mixed-species aquatic weed fertilizer formulations consistently outperformed single-species formulations, while Vallisneria-based fertilizers underperformed. Application dose and preparation form exerted secondary or negligible effects. Growth enhancement was expressed primarily through increased shoot biomass and canopy development, with root biomass, shoot–root allocation, and leaf chlorophyll status remaining largely conserved. These results indicate that combining multiple submerged macrophyte species into a single fertilizer formulation enhances nutrient availability and utilization efficiency rather than increasing total nutrient input. 4.1 Synergistic effects of mixed aquatic weed-species fertilizers on plant growth The consistently high and dose-stable performance of the mixed-species fertilizer indicates the presence of non-additive mixture effects, widely reported in plant residue and litter decomposition studies. When chemically and structurally diverse plant materials are combined, decomposition dynamics often deviate from the average behavior of individual components due to complementary interactions among substrates and decomposer communities (Gartner and Cardon 2004 ; Hättenschwiler et al. 2005 ). In the present system, combining three submerged macrophyte species with organic co-amendments generated a fertilizer matrix that promoted robust crop growth under greenhouse conditions. Previous studies have shown that Egeria densa , Elodea nuttallii , and Vallisneria sp. differ in nutrient concentrations, tissue structure, and organic matter quality, particularly with respect to nitrogen, phosphorus, potassium, and decomposition dynamics (Sampaio and Oliveira 2005 ; Xiao et al. 2009 ; Muñoz Escobar et al. 2011 ; Zoppi et al. 2024 ). Mixing these species biomasses likely produced a fertilizer with a more balanced nutrient profile and a wider range of organic compounds. Such diversity likely supported complementary microbial activity and buffered nutrient release, reducing nutrient bottlenecks associated with chemically homogeneous residues. The robustness of mixed-species fertilizer performance across application doses further supports this interpretation. Whereas single-species fertilizers exhibited dose-dependent responses, particularly for shoot biomass, the mixed formulation maintained high productivity even at lower application rates. This buffering effect is consistent with findings from multi-feedstock organic fertilizer systems, which often exhibit greater nutrient-use efficiency and stability than single-feedstock inputs (Martínez-Nieto et al. 2011 ; Lizundia et al. 2022 ; Dissanayaka et al. 2023 ). Notably, these effects occurred without increased root biomass investment, indicating sufficient nutrient availability to sustain shoot growth without inducing compensatory belowground allocation. 4.2 Underperformance of Vallisneria-based fertilizer Among the single-species formulations, Vallisneria-based fertilizer consistently resulted in lower biomass production and weaker morphological responses compared with Egeria- and Elodea-based treatments. Two complementary mechanisms likely contributed to this pattern. First, Vallisneria biomass contains lower nitrogen concentrations on a dry-weight basis than the other species used, potentially limiting early vegetative growth in short-cycle leafy crops such as Brassica rapa . Second, interspecific differences in tissue structure and biochemical composition can influence mineralization rates, with more recalcitrant residues releasing nutrients more slowly and reducing synchrony between nutrient availability and crop demand (Freschet et al. 2012 ). The limited responsiveness of Vallisneria-based fertilizer to increasing application rates supports this interpretation, as higher quantities did not compensate for less favorable nutrient release dynamics. While direct nutrient profiling and decomposition assays were beyond the scope of this experiment, the consistency of Vallisneria underperformance across multiple growth metrics suggests that residue quality and mineralization timing were key drivers of its reduced agronomic effectiveness. 4.3 Shoot-driven growth responses and conserved biomass allocation Across all treatments, increases in total plant biomass were driven primarily by enhanced shoot growth, while root biomass and shoot–root allocation ratios remained largely unchanged. This pattern indicates that aquatic weed–based fertilizers improved aboveground productivity without inducing compensatory belowground investment, consistent with efficient nutrient uptake and utilization (Poorter et al. 2012 ). In nutrient-limited conditions, plants often allocate proportionally more resources to root growth; the absence of such a response here implies that nutrient availability was sufficient to support shoot expansion throughout the growth period. The mixed-species fertilizer, in particular, promoted canopy development through increased leaf number and plant height, reflecting improved nutrient supply that supported leaf initiation and expansion rather than altering fundamental allocation strategies. Similar shoot-driven growth responses have been reported for Brassica rapa under organic fertilization regimes where nutrient release is well synchronized with crop demand (Tamai et al. 2023 ; Zhang et al. 2024 ). 4.4 Stable chlorophyll status despite increased biomass production Despite pronounced differences in biomass and morphology, leaf chlorophyll index (SPAD) remained relatively stable across treatments. This indicates that fertilizer-induced growth enhancement was not driven by increased chlorophyll concentration per unit leaf area but by expansion of total photosynthetically active tissue. SPAD values typically plateau once leaf nitrogen sufficiency is achieved, particularly in fast-growing leafy vegetables, such that additional nutrient supply primarily promotes leaf area development rather than pigment accumulation (Wood et al. 1993 ; Padilla et al. 2018 ). The stable SPAD values observed here, alongside strong increases in shoot biomass and leaf production, are therefore consistent with structural growth driven by adequate nutrient availability rather than shifts in leaf nitrogen status. This highlights the importance of integrating both physiological indices and morphological traits when evaluating organic fertilizer performance. 4.5 Limited influence of application form Application form (raw solid versus liquid extract) exerted only a minor influence on plant growth compared with fertilizer composition and dose. Although liquid organic fertilizers are often assumed to enhance crop performance through more rapid nutrient solubilization, the absence of significant differences between application forms in this study indicates that nutrient release from the solid aquatic weed–based fertilizer matrix was sufficient to meet crop demand under the experimental conditions. Several factors may explain this limited form effect. The aquatic weed biomass and associated co-amendments were finely processed and incorporated into soil, likely facilitating rapid microbial colonization and mineralization following application. Consequently, nutrient availability from the raw solid form may have closely matched that of the liquid extract during the active growth phase of Brassica rapa . In addition, both application forms delivered equivalent total fertilizer inputs, potentially leading to convergence in cumulative plant-available nutrient pools over time. From a practical perspective, this finding implies that labor- and energy-intensive extraction processes are not strictly necessary to achieve effective fertilization when using aquatic weed biomass under controlled conditions. However, application form may become more influential in systems characterized by greater environmental variability, nutrient losses, or extended cropping durations. In such contexts, the gradual nutrient release associated with solid organic fertilizers may be advantageous for long-cycle crops, agroforestry systems, or forest regeneration, where sustained nutrient supply is desirable. Further research integrating direct measurements of nutrient mineralization dynamics and field-scale validation across diverse crops and environments will be necessary to determine the conditions under which liquid formulations provide clear agronomic benefits (Baghdadi et al. 2018 ; Poveda 2022 ). 4.6 Implications for circular nutrient recovery, aquatic weed management, and environmental sustainability The findings of this study have direct relevance for circular nutrient management and freshwater ecosystem management strategies. Submerged aquatic weeds are widely treated as problematic biomass requiring costly removal to maintain ecosystem function. Demonstrating that mixed-species aquatic weed biomass can be converted into an effective organic fertilizer reframes aquatic weed management from waste disposal to nutrient recovery, aligning freshwater maintenance with agricultural productivity. The integration of locally available organic residues enhances the applicability of this approach, reflecting realistic organic fertilization systems in which co-amendments support microbial activity and nutrient cycling, particularly in low-input and resource-constrained contexts. Mixed-species formulations may also buffer variability in species composition and biomass availability, increasing the resilience of nutrient recycling strategies under climate and management uncertainty. While this study focused on a short-cycle leafy vegetable under greenhouse conditions, the principles of residue diversity, nutrient complementarity, and buffered release are broadly applicable and warrant validation across crops, soils, and field environments. 5 Conclusions This study demonstrates that submerged aquatic weeds can be effectively valorized as organic fertilizers within circular nutrient recovery systems, with fertilizer identity emerging as the dominant determinant of crop performance. Across all measured growth, morphological, and physiological parameters, mixed-species aquatic weed fertilizer consistently outperf ormed single-species formulations, while application dose and preparation form exerted secondary or negligible effects. Growth enhancement was primarily driven by increased shoot biomass and canopy development, with root biomass allocation and leaf chlorophyll status remaining largely conserved. The superior and dose-stable performance of mixed-species formulations indicates the presence of synergistic effects arising from residue diversity, likely mediated through complementary nutrient profiles and buffered nutrient release dynamics. Rather than increasing total nutrient input, mixed-species fertilizers appear to enhance the efficiency and temporal synchronization of nutrient availability, supporting structural growth without inducing compensatory belowground investment. These findings align with broader ecological and agronomic evidence that chemically diverse organic substrates can generate non-additive benefits for nutrient cycling and plant productivity. From a systems perspective, the results highlight the potential of mixed-species aquatic weed fertilizers to bridge freshwater ecosystem management and sustainable agriculture. By transforming problematic submerged macrophyte biomass and locally available organic residues into effective growth-promoting inputs, this approach reframes aquatic weed removal from a waste management challenge into a resource recovery opportunity. Such strategies are particularly relevant under climate change, where resilient, low-input, and locally adaptable nutrient management solutions are increasingly needed. While this study was conducted under controlled greenhouse conditions using a short-cycle leafy vegetable, the underlying principles of residue diversity, nutrient complementarity, and buffered release are broadly applicable. Future research integrating direct nutrient profiling, decomposition dynamics, and field-scale validation across diverse crops and environments will be essential to fully realize the potential of aquatic weed-based fertilizers within climate-responsive and circular agricultural systems. Abbreviations SDGs Sustainable Development Goals Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests Funding This research did not receive funding. Author Contribution CMAA and NY designed the experiment. CMAA collected and analyzed the data and drafted the manuscript. NY supervised the research and critically reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. Data Availability All data supporting the findings of this study are available within the paper and its supplementary material. References Abbas, M., Atiq-ur-Rahman, M., Manzoor, F. & Farooq, A. 03. A quantitative analysis and comparison of nitrogen, potassium and phosphorus in rice husk and wheat bran samples. Pure Appl. Biology (PAB) . 1 , 14–15 (2012). Alauddin, M., Mohison, G. M., Ali, A. H. M. Z. & Rahman, M. K. Interactive effects of rice bran compost and chemical fertilizers on macronutrients, oil and protein content in sunflower (Helianthus annuus L.). International Journal of Agricultural Research, Innovation and Technology (IJARIT). (2020). https://doi.org/10.22004/ag.econ.309456 Asaeda, T., Senavirathna, M. D. H. J. & Vamsi Krishna, L. Evaluation of Habitat Preferences of Invasive Macrophyte Egeria densa in Different Channel Slopes Using Hydrogen Peroxide as an Indicator. Front. Plant. 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Decomposition dynamics in mixed-species leaf litter. Oikos 104 , 230–246. https://doi.org/10.1111/j.0030-1299.2004.12738.x (2004). Hättenschwiler, S., Tiunov, A. V. & Scheu, S. Biodiversity and Litter Decomposition in Terrestrial Ecosystems. Annu. Rev. Ecol. Evol. Syst. 36 , 191–218. https://doi.org/10.1146/annurev.ecolsys.36.112904.151932 (2005). IPCC. AR6 Synthesis Report: Climate Change 2023. (2023). https://www.ipcc.ch/report/ar6/syr/ . Accessed 27 Dec 2025. Kang, M-Y. et al. Effects of Rice Bran Application on Growth, Yield, and Palatability of Rice. KOREAN J. CROP Sci. 53 , 24–30 (2008). Khanna, M., Zilberman, D., Hochman, G. & Basso, B. An economic perspective of the circular bioeconomy in the food and agricultural sector. Commun. Earth Environ. 5 , 507. https://doi.org/10.1038/s43247-024-01663-6 (2024). Kumar, A., Thakur, M. K., Hart, P. & Thakur, V. K. Sustainable Valorization of Spent Coffee Grounds: A Green Chemistry Approach to Soil Amendment and Environmental Monitoring. ACS Sustainable Resour. Manage. 2 , 1630–1642. https://doi.org/10.1021/acssusresmgt.5c00083 (2025). Li, H. et al. Long-term oyster shell powder applications increase crop yields and control soil acidity and cadmium in red soil drylands. Front. Plant. Sci. 16 , 1506733. https://doi.org/10.3389/fpls.2025.1506733 (2025). Lizundia, E., Luzi, F. & Puglia, D. Organic waste valorisation towards circular and sustainable biocomposites. Green. Chem. 24 , 5429–5459. https://doi.org/10.1039/D2GC01668K (2022). Ma, D. et al. Synergy of Biochar and Organic Fertilizer Reduces Phosphorus Leaching. Agronomy 15 , 2528. https://doi.org/10.3390/agronomy15112528 (2025). Martínez-Nieto, P., Bernal-Castillo, J., Calixto-Díaz, M. & BIOFERTILIZERS AND COMPOSTING ACCELERATORS OF POLLUTING MACROPHYTES OF A COLOMBIAN LAKE. J. soil. Sci. plant. Nutr. 11 :47–61. https://doi.org/10.4067/S0718-95162011000200005 (2011). Muñoz Escobar, M., Voyevoda, M., Fühner, C. & Zehnsdorf, A. Potential uses of Elodea nuttallii-harvested biomass. Energ. Sustain. Soc. 1 , 4. https://doi.org/10.1186/2192-0567-1-4 (2011). Oki, Y. & Une, K. Relationship between occurrence of aquatic weeds and water quality in natural water bodies (3). Weed Res. Japan . 34 (Suppl.), 97–98 (1989). (In Japanese). Padilla, F. M. et al. Different Responses of Various Chlorophyll Meters to Increasing Nitrogen Supply in Sweet Pepper. Front. Plant. Sci. 9 , 1752. https://doi.org/10.3389/fpls.2018.01752 (2018). Pal, P. et al. Circular Bioeconomy in Action: Transforming Food Wastes into Renewable Food Resources. Foods 13 , 3007. https://doi.org/10.3390/foods13183007 (2024). Poorter, H. et al. Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control. New Phytol. 193 , 30–50. https://doi.org/10.1111/j.1469-8137.2011.03952.x (2012). Porre, R. J. et al. Is litter decomposition enhanced in species mixtures? A meta-analysis. Soil Biol. Biochem. 145 , 107791. https://doi.org/10.1016/j.soilbio.2020.107791 (2020). Poveda, J. The use of freshwater macrophytes as a resource in sustainable agriculture. J. Clean. Prod. 369 , 133247. https://doi.org/10.1016/j.jclepro.2022.133247 (2022). Rockström, J. et al. A safe operating space for humanity. Nature 461 , 472–475. https://doi.org/10.1038/461472a (2009). Sampaio, E. V. S. B. & Oliveira, N. M. B. Aproveitamento da macrófita aquática Egeria densa como adubo orgânico. Planta daninha . 23 , 169–174. https://doi.org/10.1590/S0100-83582005000200001 (2005). Tamai, T., Yoshimura, D., Yamaguchi, M. & Wakita, K. Effects of waterweed compost derived from Lake Biwa on Komatsuna (Brassica rapa var. perviridis) growth. Plant. Prod. Sci. 26 , 143–152. https://doi.org/10.1080/1343943X.2023.2197242 (2023). United Nations. Transforming our world: the 2030 Agenda for Sustainable Development. (2015). https://sdgs.un.org/2030agenda . Accessed 27 Dec 2025. Wood, C. W., Reeves, D. W. & Himelrick, D. G. Relationships between chlorophyll meter readings and leaf chlorophyll concentration, N status, and crop yield: A review. Proceedings Agronomy Society of NZ 23: (1993). Xiao, L. et al. Solid state fermentation of aquatic macrophytes for crude protein extraction. Ecol. Eng. 35 , 1668–1676. https://doi.org/10.1016/j.ecoleng.2008.08.004 (2009). Zhang, M. et al. Application of oyster shell powder for five consecutive years effectively controlled soil acidification and reduced cadmium accumulation in rice grains. Sci. Rep. 15 , 12008. https://doi.org/10.1038/s41598-025-96700-5 (2025). Zhang, Y. et al. Effects of the Application of Organic Fertilizers on the Yield, Quality, and Soil Properties of Open-Field Chinese Cabbage (Brassica rapa spp. pekinensis) in China: A Meta-Analysis. Agronomy 14 , 2555. https://doi.org/10.3390/agronomy14112555 (2024). Zoppi, M., Falasco, E., Schoefs, B. & Bona, F. Turning waste into resources: A comprehensive review on the valorisation of Elodea nuttallii biomass. J. Environ. Manage. 369 , 122258. https://doi.org/10.1016/j.jenvman.2024.122258 (2024). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 May, 2026 Reviews received at journal 14 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 29 Apr, 2026 Editor invited by journal 11 Feb, 2026 Editor assigned by journal 06 Feb, 2026 Submission checks completed at journal 06 Feb, 2026 First submitted to journal 05 Feb, 2026 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. 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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-8796299","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":635239577,"identity":"1e8ebd76-cee1-4e16-b033-4397d5c6ade0","order_by":0,"name":"Cedric Mankponse Antoine Assogba","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYNACAyiZwCAhB2IdeIBfPWMDkhYLY7CWBIJaEHZVJIJ5+LTwt589/uBDwR05/vbmbR8e1Eikzw87/BBoi52cbgN2LRJn8hIbZxg8M5Y4c6x4RsIxidyNt9MMgFqSjc0O4LDmQI5hM4/B4cQNEjnGDIkNQC2zE0BaDiRuw6FF/vwbsJb6DfJvwFrSDWenf8CrxeAGxJYEAwkesJYEeekc/LYY3nhjOHOGwWHDGWfSihmAfjHcIJ1TcCDBALdf5M7nGHz48OewPH/74c2MP2rq5OVnp2/+8KHCTg6n9zGdClZpQKxyEJBvIEX1KBgFo2AUjAQAAIR4Y+Tw5AgcAAAAAElFTkSuQmCC","orcid":"","institution":"Okayama University","correspondingAuthor":true,"prefix":"","firstName":"Cedric","middleName":"Mankponse Antoine","lastName":"Assogba","suffix":""},{"id":635239578,"identity":"5547e81c-0ab7-42b6-8e4a-062ea5fd77e7","order_by":1,"name":"Nakashima Yoshitaka","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Nakashima","middleName":"","lastName":"Yoshitaka","suffix":""}],"badges":[],"createdAt":"2026-02-05 11:26:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8796299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8796299/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108689825,"identity":"748cbb81-ceae-4b94-9ca5-6318ba00060b","added_by":"auto","created_at":"2026-05-07 10:42:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1318154,"visible":true,"origin":"","legend":"\u003cp\u003eVisual overview of plant responses to aquatic-weed–based fertilizer treatments prior to harvest.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/41d40eea99c24cd204ff449e.png"},{"id":108689758,"identity":"ead32578-4d43-4090-ba4e-1dc74c79214c","added_by":"auto","created_at":"2026-05-07 10:42:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97404,"visible":true,"origin":"","legend":"\u003cp\u003eTotal fresh biomass of \u003cem\u003eBrassica rapa\u003c/em\u003e per plant across fertilizer types and application doses. Boxes represent interquartile ranges with median lines and points represent individual observations. Different letters denote significant differences among fertilizer types (Tukey HSD, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/ccdb1fd462200d7e384c33d4.png"},{"id":108689699,"identity":"d70dac8a-3da4-4409-841d-79801124b871","added_by":"auto","created_at":"2026-05-07 10:42:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83560,"visible":true,"origin":"","legend":"\u003cp\u003eTotal fresh biomass of \u003cem\u003eBrassica rapa\u003c/em\u003e per plant across fertilizer types and application doses. Boxes represent interquartile ranges with median lines and points represent individual observations. Different letters denote significant differences among fertilizer types (Tukey HSD, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/a36420584f312abc1edd71a4.png"},{"id":108689749,"identity":"1c0646a0-0ce6-453b-8cb9-98e000d1da74","added_by":"auto","created_at":"2026-05-07 10:42:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53086,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction effects of fertilizer type and dose on shoot biomass. Points represent estimated marginal means and error bars indicate ± SE (n=4).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/800f303f1255d784ba278a7a.png"},{"id":108689725,"identity":"339f1a01-ed18-4c54-a4a8-424dc1982e15","added_by":"auto","created_at":"2026-05-07 10:42:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87416,"visible":true,"origin":"","legend":"\u003cp\u003ePlant height responses of \u003cem\u003eBrassica rapa\u003c/em\u003e to aquatic-weed–based fertilizers across fertilizer types and application doses. Values represent mean ± SE (n = 4) and points represent individual observations. Letters indicate significant differences among fertilizer types within each dose level (Tukey HSD, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/61994d568dd4765f1303d838.png"},{"id":108689721,"identity":"4e2c835a-fcc1-4ee2-b2ce-e9928e57f079","added_by":"auto","created_at":"2026-05-07 10:42:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":73321,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf production responses of \u003cem\u003eBrassica rapa\u003c/em\u003e to aquatic-weed–based fertilizers across fertilizer types and application doses. Values represent mean ± SE (n = 4) and points represent individual observations. Letters indicate significant differences among fertilizer types within each dose level (Tukey HSD, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/dfccfcc3378e013aad02e4b1.png"},{"id":108805556,"identity":"a896e67d-4685-4a31-9a42-00168a6ef0b2","added_by":"auto","created_at":"2026-05-08 15:26:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2464778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/3544ceee-5639-4058-9a72-d5ce2c730c21.pdf"},{"id":108689730,"identity":"b9eaaa77-869d-4ba3-87ff-86d6b630d9cc","added_by":"auto","created_at":"2026-05-07 10:42:08","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":60544,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8796299/v1/b0d57fd1f2998df411c9b6e6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mixed-species aquatic weed fertilizers enhance Brassica rapa growth through nutrient synergy within circular nutrient recovery systems","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlobal food production systems are increasingly constrained by climate change, nutrient pollution, and accelerating degradation of terrestrial and freshwater ecosystems. These pressures compromise nutrient cycling efficiency, freshwater ecosystem functioning, and long-term agricultural productivity, while intensifying trade-offs between food security and environmental protection (Rockstr\u0026ouml;m et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; FAO 2017; IPCC \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Addressing these interconnected challenges requires nutrient management strategies that simultaneously support crop production, reduce environmental burdens, and enhance resilience under climate variability, directly contributing to multiple Sustainable Development Goals (United Nations \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSubmerged aquatic weeds (macrophytes) represent a persistent environmental management challenge in freshwater ecosystems worldwide, particularly under conditions of nutrient enrichment and altered hydrology. In Japan and many temperate regions, species such as \u003cem\u003eEgeria densa\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eVallisneria sp.\u003c/em\u003e proliferate rapidly in eutrophic waters, forming dense stands that obstruct waterways, disrupt hydrological processes, reduce dissolved oxygen availability, and displace native aquatic biodiversity. Management typically relies on mechanical harvesting, which is costly, recurrent, and often addresses symptoms rather than underlying nutrient drivers of weed proliferation (Asaeda et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a result, large quantities of harvested aquatic biomass are commonly treated as waste despite their substantial accumulation of nutrients and organic matter (Zoppi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom a circular nutrient management perspective, submerged aquatic weed biomass represents a largely untapped resource. Aquatic macrophytes actively assimilate nitrogen, phosphorus, potassium, and micronutrients from water and sediments, functioning as effective nutrient sinks in freshwater systems (Mu\u0026ntilde;oz Escobar et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zoppi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Their removal therefore intersects directly with eutrophication mitigation and nutrient recovery, processes expected to become increasingly important under climate change scenarios characterized by intensified nutrient loading and hydrological variability (Dissanayaka et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, organic fertilizer systems frequently incorporate co-amendments derived from agricultural by-products, food processing residues, and mineral sources to stimulate microbial activity, enhance nutrient retention, and stabilize soil chemical conditions (Lizundia et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cereal by-products such as rice bran and food residues such as spent coffee grounds have been shown to improve nutrient availability and crop performance through their effects on microbial processes and gradual nutrient release, while calcium-rich materials such as oyster shell powder contribute to pH buffering and nutrient balance (Kang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Alauddin et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Birnbaum et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Integrating submerged aquatic weed biomass within such composite organic matrices reflects realistic fertilization practices and allows the functional role of aquatic biomass to be evaluated within operationally relevant nutrient systems.\u003c/p\u003e \u003cp\u003eIn parallel, although aquatic weed biomass has attracted increasing attention as a potential organic fertilizer or compost feedstock, existing studies have largely focused on single-species applications or nutrient characterization, with limited emphasis on mixed-species biomass applications or composts. This contrasts with real-world harvesting conditions, where aquatic weed biomass typically consists of mixed-species assemblages rather than monospecific stands. Evidence from litter decomposition and organic amendment research demonstrates that mixing chemically and structurally diverse plant residues can generate non-additive, synergistic effects on decomposition rates, nutrient mineralization, and plant-available nutrient pools. These effects arise from complementary nutrient profiles, heterogeneous substrates, and enhanced microbial interactions, resulting in more balanced and temporally buffered nutrient release (Gartner and Cardon \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; H\u0026auml;ttenschwiler et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Porre et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite this well-established ecological foundation, the agronomic implications of mixed-species aquatic weed fertilizers, particularly in short-cycle vegetable systems, remain poorly understood. Moreover, the relative importance of fertilizer composition relative to application dose and preparation form has not been systematically resolved.\u003c/p\u003e \u003cp\u003eAddressing these knowledge gaps is particularly relevant in the context of climate-responsive and circular-economy-oriented nutrient management strategies, where resilience, efficiency, and adaptability of organic inputs are increasingly valued (Khanna et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mixed-species organic fertilizers may offer greater buffering capacity against variability in biomass availability, nutrient composition, and environmental conditions, thereby enhancing the robustness of nutrient recycling systems. If effective, such approaches could simultaneously support freshwater ecosystem management, reduce organic waste disposal pressures, improve nutrient-use efficiency, and contribute to sustainable crop production under increasing climatic uncertainty.\u003c/p\u003e \u003cp\u003eAgainst this background, the present study evaluated the agronomic performance of fertilizers derived from submerged aquatic weeds \u003cem\u003eEgeria densa\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eVallisneria sp.\u003c/em\u003e, applied as single-species or mixed-species formulations and combined with locally available organic co-amendments. Using \u003cem\u003eBrassica rapa\u003c/em\u003e L. var. \u003cem\u003eperviridis\u003c/em\u003e as a model short-cycle leafy vegetable, this study assessed the effects of fertilizer type, application dose, and application form on plant biomass production, morphological development, biomass allocation, and physiological status under controlled greenhouse conditions. By linking aquatic weed valorization with crop productivity, this study provides experimental evidence supporting circular nutrient recovery strategies at the interface of freshwater ecosystem management, sustainable agriculture, and climate-responsive environmental stewardship.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Aquatic weed biomass sources and nutrient profiles\u003c/h2\u003e \u003cp\u003eBiomass of three submerged macrophyte species commonly occurring in Japanese freshwater ecosystems, \u003cem\u003eEgeria densa\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eVallisneria sp\u003c/em\u003e., was collected from waterways in Urayasu, Okayama Prefecture, Japan, in January 2025 (Table S0.1). Collected biomass was rinsed with tap water to remove adhering sediments and debris, oven-dried at 60\u0026deg;C to constant weight, and ground using a Wonder Blender WB-1 from Osaka Chemical Co. Ltd. prior to fertilizer preparation.\u003c/p\u003e \u003cp\u003eBaseline nutrient composition data for each macrophyte species and organic residues, including nitrogen (N), phosphorus (P), and potassium (K) were obtained from published literature. These data were used to characterize relative nutrient profiles among species and to inform fertilizer formulation and component ratios. A summary of the nutrient characteristics of each species and organic residues is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Total N, P, and K are expressed on an average percentage dry weight basis.\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\u003eNutrient composition of submerged macrophyte species and organic inputs used for fertilizer formulation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Oki and Une, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1989\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttalli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Oki and Une, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1989\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVallisneria natans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Oki and Une, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1989\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRice husk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Abbas et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpent coffee grounds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Birnbaum et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Fertilizer formulation and preparation\u003c/h2\u003e \u003cp\u003eAll fertilizer treatments were prepared using a standardized organic base mixture to ensure comparability across treatments. The master dry fertilizer mix consisted of 65% aquatic weed biomass, 15% rice bran, 15% spent coffee grounds, and 5% oyster shell powder (w/w). The aquatic biomass fraction constituted the primary nutrient source for the different fertilizer treatments and represented the experimental factor of interest. Rice bran and spent coffee grounds were included as readily available organic co-substrates to enhance microbial activity and nutrient mineralization, while oyster shell powder was incorporated at a low proportion as a calcium source and pH-buffering agent. The ratio was selected to balance nutrient supply, decomposition rate, and material stability while avoiding excessive alkalinity or nutrient immobilization.\u003c/p\u003e \u003cp\u003eFor single-species fertilizers, the aquatic biomass fraction comprised exclusively one macrophyte species (\u003cem\u003eE. densa\u003c/em\u003e, \u003cem\u003eE. nuttallii\u003c/em\u003e, or \u003cem\u003eVallisneria sp.\u003c/em\u003e). For the mixed-species fertilizer, the biomass fraction was composed of equal proportions of the three species. Rice bran and coffee grounds were air-dried prior to mixing, and all components were thoroughly homogenized before application.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental design and treatment structure\u003c/h2\u003e \u003cp\u003eThe experiment followed a completely randomized design (CRD) with four replicates per treatment, yielding a total of 104 experimental units (26 treatments \u0026times; 4 replicates), each consisting of one plant.\u003c/p\u003e \u003cp\u003eTwenty-four treatments were arranged in a 4 \u0026times; 3 \u0026times; 2 factorial structure combining four fertilizer types, three application doses, and two application forms. Fertilizer types were coded as ED (\u003cem\u003eEgeria densa\u003c/em\u003e), EN (\u003cem\u003eElodea nuttallii\u003c/em\u003e), VA (\u003cem\u003eVallisneria sp.\u003c/em\u003e), and MX (\u003cem\u003emixed species\u003c/em\u003e); dose levels as low (L), optimum (O), and high (H); and application forms as raw solid (R) or liquid extract (E). Two additional controls were included: an unfertilized control (CTRL) and a standard commercial control (COMM) consisting of a pre-fertilized seed-sowing soil commonly used for leafy vegetable cultivation in Okayama. Treatment allocation was fully randomized at the start of the experiment, and the randomized arrangement was maintained after transfer from the incubator to the greenhouse. Treatment codes and descriptions are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of experiment treatments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment label\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFertilizer identity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDose level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eApplication form\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTRL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUnfertilized control\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCOMM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStandard commercial control\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eED-L-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEgeria-based fertilizer, low dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eED-O-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEgeria-based fertilizer, optimum dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eED-H-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEgeria-based fertilizer, high dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eED-L-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEgeria-based fertilizer, low dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eED-O-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEgeria-based fertilizer, optimum dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eED-H-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEgeria densa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEgeria-based fertilizer, high dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEN-L-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttallii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElodea-based fertilizer, low dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEN-O-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttallii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElodea-based fertilizer, optimum dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEN-H-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttallii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElodea-based fertilizer, high dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEN-L-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttallii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElodea-based fertilizer, low dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEN-O-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttallii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElodea-based fertilizer, optimum dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEN-H-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eElodea nuttallii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eElodea-based fertilizer, high dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVA-L-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVallisneria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVallisneria-based fertilizer, low dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVA-O-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVallisneria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVallisneria-based fertilizer, optimum dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVA-H-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVallisneria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVallisneria-based fertilizer, high dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVA-L-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVallisneria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVallisneria-based fertilizer, low dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVA-O-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVallisneria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVallisneria-based fertilizer, optimum dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVA-H-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVallisneria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVallisneria-based fertilizer, high dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMX-L-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMixed species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMixed-species fertilizer, low dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMX-O-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMixed species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMixed-species fertilizer, optimum dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMX-H-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMixed species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRaw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMixed-species fertilizer, high dose, raw solid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMX-L-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMixed species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMixed-species fertilizer, low dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMX-O-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMixed species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMixed-species fertilizer, optimum dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMX-H-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMixed species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMixed-species fertilizer, high dose, liquid extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Soil substrate and pot preparation\u003c/h2\u003e \u003cp\u003eField soil was collected from the Okayama University Field Research Center, air-dried, and sieved to remove coarse debris. During the incubator phase, each pot was filled with 300 g of air-dried soil and fitted with drainage holes to prevent waterlogging.\u003c/p\u003e \u003cp\u003eAfter transplanting to larger pots for the greenhouse phase, each pot was filled with approximately 1.52 kg of the same soil to provide increased root volume while maintaining consistency in soil properties across growth stages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fertilizer dosage levels and application forms\u003c/h2\u003e \u003cp\u003eEach fertilizer type was applied at three dosage levels based on soil dry weight: low (0.5%, 1.5 g per pot), optimum (1.0%, 3.0 g per pot), and high (2.0%, 6.0 g per pot). Fertilizers were applied either as raw solid material or as a liquid extract prepared to deliver an equivalent total fertilizer mass per pot.\u003c/p\u003e \u003cp\u003eLiquid fertilizer extracts were prepared by mixing the required fertilizer mass with distilled water at a 1:10 (w/v) ratio, heating at 95\u0026deg;C for 30 min, cooling to room temperature, and filtering to remove solids. Fresh extracts were prepared prior to each application to minimize microbial alteration of nutrient composition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plant material and growth conditions\u003c/h2\u003e \u003cp\u003eSeeds of \u003cem\u003eB. rapa\u003c/em\u003e var. \u003cem\u003eperviridis\u003c/em\u003e were surface-sterilized and germinated under controlled conditions using a petri dish. Uniform seedlings were transplanted 6 days after gemination at the one-true-leaf stage into prepared pots.\u003c/p\u003e \u003cp\u003eFor the first 35 days, plants were grown in an incubator to ensure uniform early establishment, using V-shaped plastic nursery pots (height 7 cm, bottom diameter 6 cm, top diameter 9 cm). Incubator conditions were maintained at 25\u0026deg;C during the light period and 20\u0026deg;C during the dark period under a 12 h light: 12 h dark photoperiod. Raw fertilizer treatments were applied once as a top-dressing 7 days after germination. In contrast, liquid fertilizer extract treatments were applied twice weekly (every 3 days) for approximately four weeks, starting on day 7 after germination, resulting in seven applications in total. For extract treatments, the assigned fertilizer dose was divided evenly among applications.\u003c/p\u003e \u003cp\u003eFrom day 36, all treatments per pot were transplanted into larger V-shaped plastic nursery pots (height 12 cm, bottom diameter 10 cm, top diameter 15 cm) and transferred to a greenhouse. Plants were grown under natural light and temperature conditions until harvest on day 63. The total experiment run from 18 February to 22 April 2025.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Growth measurements\u003c/h2\u003e \u003cp\u003ePlant height, leaf number, and chlorophyll index (SPAD) were measured weekly throughout the experiment. At harvest, shoot and root fresh weight were recorded, followed by oven-drying at 60\u0026deg;C to constant weight to determine shoot and root dry biomass.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eData analysis followed a two-step approach reflecting the experimental structure. First, a three-way factorial analysis of variance (ANOVA) was conducted on the 24 factorial treatments to assess the effects of fertilizer type, application dose, application form, and their interactions. Second, a one-way ANOVA including all 26 treatments was performed to compare fertilizer treatments with the unfertilized and commercial controls.\u003c/p\u003e \u003cp\u003ePost-hoc comparisons were performed using Tukey\u0026rsquo;s honest significant difference (HSD) test, and selected contrasts against control treatments were examined using Dunnett-type comparisons. Model assumptions were evaluated using residual diagnostics, and response variables violating normality were log-transformed prior to analysis. All statistical analyses were conducted using R software (version 4.5.2).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Overall plant responses to aquatic-weed\u0026ndash;based fertilizers\u003c/h2\u003e \u003cp\u003eAcross all measured variables, including biomass production, plant height, leaf number, and chlorophyll index (SPAD), clear differences in plant growth were observed among fertilizer treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Visual inspection prior to harvest indicated marked variation in aboveground development across fertilizer types, with mixed-species (MX) treatments exhibiting more uniform canopy development than single-species treatments, particularly those based on \u003cem\u003eVallisneria sp.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects of fertilizer treatments on total plant biomass\u003c/h2\u003e \u003cp\u003eTotal plant biomass responded significantly to fertilizer identity (type). Factorial ANOVA revealed a significant main effect of fertilizer type on both total fresh weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0037) and total dry weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019), whereas dose, application form, and interaction terms were not significant (Tables S1.1 and S1.2). Across all treatments, the mixed-species fertilizer (MX) produced the highest total biomass, followed by \u003cem\u003eElodea nuttallii\u003c/em\u003e\u0026ndash;based (EN) and \u003cem\u003eEgeria densa\u003c/em\u003e\u0026ndash;based (ED) fertilizers, while \u003cem\u003eVallisneria sp\u003c/em\u003e\u0026ndash;based fertilizer (VA) consistently resulted in the lowest biomass accumulation.\u003c/p\u003e \u003cp\u003eWhen all 26 treatments were compared, significant differences in total fresh and dry biomass were detected (Tables S1.3 and S1.4). Several MX-based treatments and high-performing EN treatments occupied the upper end of the biomass distribution, whereas VA treatments were consistently among the lowest. Control treatments generally fell within an intermediate range, overlapping with multiple fertilizer treatments (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of fertilizer treatments on shoot biomass production\u003c/h2\u003e \u003cp\u003eShoot biomass production was strongly influenced by fertilizer composition, with consistent patterns observed for both fresh and dry biomass. Factorial ANOVA revealed fertilizer type as a significant main effect for shoot fresh weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and shoot dry weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0067), along with significant fertilizer type \u0026times; dose interactions for both variables (fresh weight: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026; dry weight: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046, Tables S2.1 and S2.2).\u003c/p\u003e \u003cp\u003eAcross all application doses, the mixed-species fertilizer (MX) consistently produced the highest shoot biomass, significantly outperforming single-species fertilizers at low and optimum doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among single-species fertilizers, \u003cem\u003eEgeria densa\u003c/em\u003e\u0026ndash;based (ED) and \u003cem\u003eElodea nuttallii\u003c/em\u003e\u0026ndash;based (EN) fertilizers exhibited moderate increases in shoot biomass at the highest application rate, whereas the \u003cem\u003eVallisneria\u003c/em\u003e sp.\u0026ndash;based fertilizer (VA) showed limited responsiveness to increasing dose. At the highest application level, differences in shoot dry biomass among MX, ED, and EN-treatments were reduced, while VA-treatments consistently produced the lowest shoot biomass.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Root biomass responses to fertilizer treatments\u003c/h2\u003e \u003cp\u003eIn contrast to aboveground biomass, root biomass showed limited responsiveness to fertilizer treatments. Factorial ANOVA detected no significant effects of fertilizer type, dose, application form, or their interactions on either root fresh or root dry biomass (Tables S3.1 and S3.2). Although one-way ANOVA across all treatments indicated significant overall differences (fresh weight \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0. 0179 and dry weight \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0167), post hoc comparisons revealed substantial overlap among treatments, including both controls (Tables S3.3 - S3.6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Biomass allocation patterns (shoot to root ratios)\u003c/h2\u003e \u003cp\u003eShoot-to-root biomass allocation remained largely conserved across fertilizer treatments. After log transformation, factorial ANOVA revealed no significant effects of fertilizer type, dose, application form, or their interactions on either fresh or dry shoot:root ratios (Tables S4.1 and S4.2). When all treatments were compared, significant variation in shoot:root fresh (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003) and dry (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0006) weight ratios was detected (Tables S4.3 and S4.4). On a fresh weight basis, several MX treatments exhibited higher shoot allocation relative to the unfertilized control. However, overall allocation patterns remained largely conserved across treatments (Tables S4.5 and S4.6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effects of aquatic-weed fertilizers on plant morphology and physiological status\u003c/h2\u003e \u003cp\u003ePlant morphological traits responded clearly to fertilizer composition. In the factorial analysis, fertilizer type exerted a significant effect on plant height (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012) and leaf number (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0082), whereas application dose and application form showed no consistent main effects. Interaction effects were marginal (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056) or non-significant (Tables S5.1 and S5.2). Across fertilizer types, the mixed-species fertilizer (MX) consistently resulted in taller plants and greater leaf production, followed by the Elodea- and Egeria-based fertilizers (EN and ED), while the Vallisneria-based fertilizer (VA) produced the smallest plants.\u003c/p\u003e \u003cp\u003eWhen all 26 treatments were compared, both plant height and leaf number differed significantly among treatments (Tables S5.3 and S5.4). Raw solid MX treatments and selected high-performing EN and liquid-extract MX treatments consistently achieved the highest plant heights, exceeding the unfertilized control (CTRL). For leaf number, low and optimum doses of raw solid MX treatments ranked highest and outperformed the commercial fertilizer control. In contrast, Vallisneria-based (VA) treatments consistently clustered among the lowest values for both traits (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast to morphological traits, leaf chlorophyll index (SPAD) remained comparatively stable across treatments. Fertilizer type, dose, and application form did not significantly affect SPAD values in the factorial analysis (Table S5.5). Although significant differences were detected when all treatments were compared (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0148), post-hoc separation was limited, and SPAD values varied within a narrow range (Tables S5.6 and S5.7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrates that aquatic weed\u0026ndash;based fertilizers derived from submerged macrophytes (\u003cem\u003eEgeria densa\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eVallisneria\u003c/em\u003e sp.) can effectively support the growth of \u003cem\u003eBrassica rapa\u003c/em\u003e, with fertilizer identity emerging as the dominant determinant of plant performance. Across biomass production, morphological development, and physiological indicators, mixed-species aquatic weed fertilizer formulations consistently outperformed single-species formulations, while Vallisneria-based fertilizers underperformed. Application dose and preparation form exerted secondary or negligible effects. Growth enhancement was expressed primarily through increased shoot biomass and canopy development, with root biomass, shoot\u0026ndash;root allocation, and leaf chlorophyll status remaining largely conserved. These results indicate that combining multiple submerged macrophyte species into a single fertilizer formulation enhances nutrient availability and utilization efficiency rather than increasing total nutrient input.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Synergistic effects of mixed aquatic weed-species fertilizers on plant growth\u003c/h2\u003e \u003cp\u003eThe consistently high and dose-stable performance of the mixed-species fertilizer indicates the presence of non-additive mixture effects, widely reported in plant residue and litter decomposition studies. When chemically and structurally diverse plant materials are combined, decomposition dynamics often deviate from the average behavior of individual components due to complementary interactions among substrates and decomposer communities (Gartner and Cardon \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; H\u0026auml;ttenschwiler et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the present system, combining three submerged macrophyte species with organic co-amendments generated a fertilizer matrix that promoted robust crop growth under greenhouse conditions.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that \u003cem\u003eEgeria densa\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eVallisneria sp.\u003c/em\u003e differ in nutrient concentrations, tissue structure, and organic matter quality, particularly with respect to nitrogen, phosphorus, potassium, and decomposition dynamics (Sampaio and Oliveira \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Xiao et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mu\u0026ntilde;oz Escobar et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zoppi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mixing these species biomasses likely produced a fertilizer with a more balanced nutrient profile and a wider range of organic compounds. Such diversity likely supported complementary microbial activity and buffered nutrient release, reducing nutrient bottlenecks associated with chemically homogeneous residues.\u003c/p\u003e \u003cp\u003eThe robustness of mixed-species fertilizer performance across application doses further supports this interpretation. Whereas single-species fertilizers exhibited dose-dependent responses, particularly for shoot biomass, the mixed formulation maintained high productivity even at lower application rates. This buffering effect is consistent with findings from multi-feedstock organic fertilizer systems, which often exhibit greater nutrient-use efficiency and stability than single-feedstock inputs (Mart\u0026iacute;nez-Nieto et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lizundia et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dissanayaka et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, these effects occurred without increased root biomass investment, indicating sufficient nutrient availability to sustain shoot growth without inducing compensatory belowground allocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Underperformance of Vallisneria-based fertilizer\u003c/h2\u003e \u003cp\u003eAmong the single-species formulations, Vallisneria-based fertilizer consistently resulted in lower biomass production and weaker morphological responses compared with Egeria- and Elodea-based treatments. Two complementary mechanisms likely contributed to this pattern. First, Vallisneria biomass contains lower nitrogen concentrations on a dry-weight basis than the other species used, potentially limiting early vegetative growth in short-cycle leafy crops such as \u003cem\u003eBrassica rapa\u003c/em\u003e. Second, interspecific differences in tissue structure and biochemical composition can influence mineralization rates, with more recalcitrant residues releasing nutrients more slowly and reducing synchrony between nutrient availability and crop demand (Freschet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe limited responsiveness of Vallisneria-based fertilizer to increasing application rates supports this interpretation, as higher quantities did not compensate for less favorable nutrient release dynamics. While direct nutrient profiling and decomposition assays were beyond the scope of this experiment, the consistency of Vallisneria underperformance across multiple growth metrics suggests that residue quality and mineralization timing were key drivers of its reduced agronomic effectiveness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Shoot-driven growth responses and conserved biomass allocation\u003c/h2\u003e \u003cp\u003eAcross all treatments, increases in total plant biomass were driven primarily by enhanced shoot growth, while root biomass and shoot\u0026ndash;root allocation ratios remained largely unchanged. This pattern indicates that aquatic weed\u0026ndash;based fertilizers improved aboveground productivity without inducing compensatory belowground investment, consistent with efficient nutrient uptake and utilization (Poorter et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In nutrient-limited conditions, plants often allocate proportionally more resources to root growth; the absence of such a response here implies that nutrient availability was sufficient to support shoot expansion throughout the growth period.\u003c/p\u003e \u003cp\u003eThe mixed-species fertilizer, in particular, promoted canopy development through increased leaf number and plant height, reflecting improved nutrient supply that supported leaf initiation and expansion rather than altering fundamental allocation strategies. Similar shoot-driven growth responses have been reported for \u003cem\u003eBrassica rapa\u003c/em\u003e under organic fertilization regimes where nutrient release is well synchronized with crop demand (Tamai et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Stable chlorophyll status despite increased biomass production\u003c/h2\u003e \u003cp\u003eDespite pronounced differences in biomass and morphology, leaf chlorophyll index (SPAD) remained relatively stable across treatments. This indicates that fertilizer-induced growth enhancement was not driven by increased chlorophyll concentration per unit leaf area but by expansion of total photosynthetically active tissue. SPAD values typically plateau once leaf nitrogen sufficiency is achieved, particularly in fast-growing leafy vegetables, such that additional nutrient supply primarily promotes leaf area development rather than pigment accumulation (Wood et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Padilla et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe stable SPAD values observed here, alongside strong increases in shoot biomass and leaf production, are therefore consistent with structural growth driven by adequate nutrient availability rather than shifts in leaf nitrogen status. This highlights the importance of integrating both physiological indices and morphological traits when evaluating organic fertilizer performance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Limited influence of application form\u003c/h2\u003e \u003cp\u003eApplication form (raw solid versus liquid extract) exerted only a minor influence on plant growth compared with fertilizer composition and dose. Although liquid organic fertilizers are often assumed to enhance crop performance through more rapid nutrient solubilization, the absence of significant differences between application forms in this study indicates that nutrient release from the solid aquatic weed\u0026ndash;based fertilizer matrix was sufficient to meet crop demand under the experimental conditions.\u003c/p\u003e \u003cp\u003eSeveral factors may explain this limited form effect. The aquatic weed biomass and associated co-amendments were finely processed and incorporated into soil, likely facilitating rapid microbial colonization and mineralization following application. Consequently, nutrient availability from the raw solid form may have closely matched that of the liquid extract during the active growth phase of \u003cem\u003eBrassica rapa\u003c/em\u003e. In addition, both application forms delivered equivalent total fertilizer inputs, potentially leading to convergence in cumulative plant-available nutrient pools over time.\u003c/p\u003e \u003cp\u003eFrom a practical perspective, this finding implies that labor- and energy-intensive extraction processes are not strictly necessary to achieve effective fertilization when using aquatic weed biomass under controlled conditions. However, application form may become more influential in systems characterized by greater environmental variability, nutrient losses, or extended cropping durations. In such contexts, the gradual nutrient release associated with solid organic fertilizers may be advantageous for long-cycle crops, agroforestry systems, or forest regeneration, where sustained nutrient supply is desirable. Further research integrating direct measurements of nutrient mineralization dynamics and field-scale validation across diverse crops and environments will be necessary to determine the conditions under which liquid formulations provide clear agronomic benefits (Baghdadi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Poveda \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Implications for circular nutrient recovery, aquatic weed management, and environmental sustainability\u003c/h2\u003e \u003cp\u003eThe findings of this study have direct relevance for circular nutrient management and freshwater ecosystem management strategies. Submerged aquatic weeds are widely treated as problematic biomass requiring costly removal to maintain ecosystem function. Demonstrating that mixed-species aquatic weed biomass can be converted into an effective organic fertilizer reframes aquatic weed management from waste disposal to nutrient recovery, aligning freshwater maintenance with agricultural productivity.\u003c/p\u003e \u003cp\u003eThe integration of locally available organic residues enhances the applicability of this approach, reflecting realistic organic fertilization systems in which co-amendments support microbial activity and nutrient cycling, particularly in low-input and resource-constrained contexts. Mixed-species formulations may also buffer variability in species composition and biomass availability, increasing the resilience of nutrient recycling strategies under climate and management uncertainty. While this study focused on a short-cycle leafy vegetable under greenhouse conditions, the principles of residue diversity, nutrient complementarity, and buffered release are broadly applicable and warrant validation across crops, soils, and field environments.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThis study demonstrates that submerged aquatic weeds can be effectively valorized as organic fertilizers within circular nutrient recovery systems, with fertilizer identity emerging as the dominant determinant of crop performance. Across all measured growth, morphological, and physiological parameters, mixed-species aquatic weed fertilizer consistently outperf ormed single-species formulations, while application dose and preparation form exerted secondary or negligible effects. Growth enhancement was primarily driven by increased shoot biomass and canopy development, with root biomass allocation and leaf chlorophyll status remaining largely conserved.\u003c/p\u003e \u003cp\u003eThe superior and dose-stable performance of mixed-species formulations indicates the presence of synergistic effects arising from residue diversity, likely mediated through complementary nutrient profiles and buffered nutrient release dynamics. Rather than increasing total nutrient input, mixed-species fertilizers appear to enhance the efficiency and temporal synchronization of nutrient availability, supporting structural growth without inducing compensatory belowground investment. These findings align with broader ecological and agronomic evidence that chemically diverse organic substrates can generate non-additive benefits for nutrient cycling and plant productivity.\u003c/p\u003e \u003cp\u003eFrom a systems perspective, the results highlight the potential of mixed-species aquatic weed fertilizers to bridge freshwater ecosystem management and sustainable agriculture. By transforming problematic submerged macrophyte biomass and locally available organic residues into effective growth-promoting inputs, this approach reframes aquatic weed removal from a waste management challenge into a resource recovery opportunity. Such strategies are particularly relevant under climate change, where resilient, low-input, and locally adaptable nutrient management solutions are increasingly needed.\u003c/p\u003e \u003cp\u003eWhile this study was conducted under controlled greenhouse conditions using a short-cycle leafy vegetable, the underlying principles of residue diversity, nutrient complementarity, and buffered release are broadly applicable. Future research integrating direct nutrient profiling, decomposition dynamics, and field-scale validation across diverse crops and environments will be essential to fully realize the potential of aquatic weed-based fertilizers within climate-responsive and circular agricultural systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSustainable Development Goals\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCMAA and NY designed the experiment. CMAA collected and analyzed the data and drafted the manuscript. NY supervised the research and critically reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas, M., Atiq-ur-Rahman, M., Manzoor, F. \u0026amp; Farooq, A. 03. A quantitative analysis and comparison of nitrogen, potassium and phosphorus in rice husk and wheat bran samples. \u003cem\u003ePure Appl. Biology (PAB)\u003c/em\u003e. \u003cb\u003e1\u003c/b\u003e, 14\u0026ndash;15 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlauddin, M., Mohison, G. M., Ali, A. H. M. Z. \u0026amp; Rahman, M. K. 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Manage.\u003c/em\u003e \u003cb\u003e369\u003c/b\u003e, 122258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jenvman.2024.122258\u003c/span\u003e\u003cspan address=\"10.1016/j.jenvman.2024.122258\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"aquatic weeds, Egeria densa, Elodea nuttalli, Vallisneria sp., waste valorization, organic fertilizer, circular bioeconomy, climate change, resource recycling, Brassica rapa L. var. perviridis","lastPublishedDoi":"10.21203/rs.3.rs-8796299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8796299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eSubmerged aquatic weeds pose persistent management challenges in freshwater ecosystems, yet their rapid biomass accumulation and nutrient-rich composition represent an underutilized resource for sustainable agriculture. Valorizing this biomass may help link freshwater ecosystem management with circular nutrient recovery and climate-resilient food systems. This study evaluated the agronomic potential of three dominant submerged macrophytes \u003cem\u003eEgeria densa\u003c/em\u003e, \u003cem\u003eElodea nuttallii\u003c/em\u003e, and \u003cem\u003eVallisneria sp\u003c/em\u003e as organic fertilizers for \u003cem\u003eBrassica rapa\u003c/em\u003e L. var. \u003cem\u003eperviridis\u003c/em\u003e, with particular emphasis on comparing single-species and mixed-species formulations integrated with locally available organic residues.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eA greenhouse experiment was conducted using a completely randomized design with 26 treatments, including four aquatic-weed\u0026ndash;based fertilizers (three single-species and one mixed-species), applied at three dosage levels and in two application forms (raw solid and liquid extract), alongside unfertilized and standard commercial controls. Fertilizer identity emerged as the primary determinant of plant performance, consistently outweighing the effects of application dose and form. Across biomass production and morphological traits, the mixed-species fertilizer produced the highest and most stable responses, particularly in shoot fresh and dry biomass, plant height, and leaf production. Single-species fertilizers showed intermediate and species-dependent performance, while \u003cem\u003eVallisneria\u003c/em\u003e-based formulations consistently resulted in lower growth. Dose effects were secondary and fertilizer-dependent, and application form had minimal influence. Growth enhancement was primarily driven by increased aboveground biomass and canopy development, while root biomass, shoot\u0026ndash;root allocation, and leaf chlorophyll index remained largely unchanged.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eThese results demonstrate that combining multiple submerged aquatic weed species into a single fertilizer formulation confers clear agronomic advantages over single-species applications. Mixed-species aquatic weed fertilizers enhance nutrient-use efficiency and crop growth without increasing application complexity, supporting their potential role in circular nutrient recovery strategies, sustainable vegetable production, and climate-resilient agricultural systems under increasing environmental variability.\u003c/p\u003e","manuscriptTitle":"Mixed-species aquatic weed fertilizers enhance Brassica rapa growth through nutrient synergy within circular nutrient recovery systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 10:40:51","doi":"10.21203/rs.3.rs-8796299/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-15T08:13:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T21:21:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201873802611840214553576303276886131611","date":"2026-05-07T07:20:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181605293123353123623743202837005777071","date":"2026-05-06T06:24:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59894227796884698741028696526008701638","date":"2026-05-04T15:18:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T12:01:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-11T11:50:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T06:25:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T06:22:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-05T10:39:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d8a04bd-bf67-43ae-bea4-f0d0cde96f54","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-15T08:13:53+00:00","index":136,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T21:21:09+00:00","index":135,"fulltext":""},{"type":"reviewerAgreed","content":"201873802611840214553576303276886131611","date":"2026-05-07T07:20:29+00:00","index":134,"fulltext":""},{"type":"reviewerAgreed","content":"181605293123353123623743202837005777071","date":"2026-05-06T06:24:16+00:00","index":133,"fulltext":""},{"type":"reviewerAgreed","content":"59894227796884698741028696526008701638","date":"2026-05-04T15:18:00+00:00","index":132,"fulltext":""},{"type":"reviewersInvited","content":"51","date":"2026-04-29T12:01:54+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67604898,"name":"Biological sciences/Ecology"},{"id":67604899,"name":"Earth and environmental sciences/Ecology"},{"id":67604900,"name":"Earth and environmental sciences/Environmental sciences"},{"id":67604901,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-07T10:40:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 10:40:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8796299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8796299","identity":"rs-8796299","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}