Abstract This study demonstrates an effective cultivation protocol for Wolffia globosa through a bio-inspired approach. Light parameters from the plant's natural, shaded habitats (PPFD ~44 µmol m⁻² s⁻¹) were first characterized to calibrate a laboratory LED system. The experiment revealed that a one-time nutrient supplementation at initiation was the critical factor for sustained growth. While all treatments were exposed to the same light regimen, the unenriched groups (C, T1, T2, and T5) experienced a "bloom-and-bust" cycle, with growth ceasing after day 9. In contrast, treatments supplemented with N-P-K fertilizer (T3 and T4) maintained a positive specific growth rate (SGR) throughout the 15-day experimental period (max SGR 0.11–0.23 day⁻¹). This finding provides a practical proof-of-concept demonstrating that a low-energy, bio-inspired lighting strategy is sufficient for cultivating W. globosa in closed systems, provided the plant's fundamental nutrient requirements are satisfied. This study presents a sustainable, energy-efficient alternative to conventional high-intensity lighting methods for aquatic plant production.

Keywords: Wolffia globosa, LED lighting, Photosynthetic photon flux density, Nutrient supplementation, Specific growth rate

Citation:  Sangmek, P., Thasnas, N., Sitthaphanit, S., and Kamolrat, N. 2026. Bio-inspired LED lighting and nutrient supplementation for sustainable Wolffia globosa cultivation. Natural and Life Sciences Communications. 25(3): e2026044.

Graphical Abstract:

INTRODUCTION

The global duckweed protein market was valued at USD 72.7 million in 2024 (Global Market Insights, 2022), highlighting the rising economic and nutritional importance of plants like Wolffia globosa Roxb. Rich in protein (up to 45%), essential amino acids, vitamins, and minerals, W. globosa is a plant with high potential as an alternative food source (Chookhampaeng et al., 2022; Said et al., 2022). Owing to these properties, W. globosa has been applied in various domains such as human food, animal feed, wastewater treatment, and bioenergy production (Kabir et al., 2010; Xie et al., 2013; Sirirustananun and Jongput, 2021).

Traditionally, W. globosa is cultivated in open ponds, which leverage its rapid growth rate to yield high biomass in nutrient-rich water (Ruekaewma et al., 2015). However, these open systems present critical limitations, including the risk of environmental contamination, uncontrolled environmental fluctuations, and inconsistent light exposure due to weather and seasonal variability (Lam et al., 2018). These factors contribute to unstable production yields, prompting a shift toward closed cultivation systems. While some studies indicate that greenhouse systems still rely primarily on natural sunlight (Kimura et al., 2023), artificial lighting, particularly energy-efficient LED systems, offers a promising solution for providing consistent and optimized light conditions (Zakurin et al., 2020; Kamolrat et al., 2023).

Despite the advantages of LEDs, there is a lack of fundamental data regarding the natural light conditions (e.g., Photosynthetic Photon Flux Density (PPFD)) in W. globosa's native habitats. Natural light's PPFD is important because it quantifies the usable light (PAR) reaching plants, directly impacting photosynthesis, growth rates and health of plants (Ge et al., 2010). While artificial lighting offers a solution for consistent cultivation, many existing protocols apply generalized high-light conditions that may not be suitable for species with specific ecological niches (Landolt and Kandeler, 1987; Petersen et al., 2022). W. globosa, unlike many open-water aquatic plants, naturally thrives in habitats shaded by surrounding vegetation (Petersen et al., 2022). This distinction is critical, as the photosynthetic apparatus of shade-adapted species is often optimized for efficient light capture at low intensities. Consequently, applying excessive light intensity typical of conventional commercial protocols not only results in unnecessary energy consumption but may also induce photoinhibition, thereby limiting growth potential, negatively affecting physiological processes and morphology (Cui and Cheng, 2015; Hazrati et al., 2016; Endo et al., 2023). However, there is a significant lack of fundamental data quantifying the precise light parameters (e.g., Photosynthetic Photon Flux Density) within these specific micro-environments.

This knowledge gap hinders the development of truly bio-inspired LED protocols tailored for W. globosa. Therefore, the primary objective of this study is two-fold: first, to characterize the authentic light conditions of Wolffia's natural, shaded habitats. Second, to replicate these "bio-inspired" light parameters in a controlled laboratory system to determine if optimizing nutrient availability can support sustained and efficient growth. This approach investigates a cultivation strategy that respects the plant's natural ecological adaptations rather than forcing it into an artificial, high-energy growth state.

MATERIALS AND METHODS

Light factors affecting W. globosa growth

Environmental light factors influencing W. globosa growth were studied by measuring light parameters in natural habitats where the species occurs. The parameters recorded included Photosynthetic Photon Flux Density (PPFD) and illuminance. These values were used to establish initial control settings for LED lighting to replicate natural light conditions conducive to optimal W. globosa growth. Subsequently, W. globosa was cultivated under LED lighting with fertilizer supplementation in tap water and compared to water sourced from natural habitats to evaluate growth performance under different cultivation conditions.

PPFD was measured in natural W. globosa habitats, which were typically shaded by large trees. Measurements were taken at midday. The collected PPFD values were then used to calibrate LED lighting in laboratory experiments to closely match natural conditions. Measurements were conducted using a spectrometer (LI-COR, model LI-180, USA).

Light illuminance was measured over a 12-hour daylight period (06:00–18:00) in both natural W. globosa habitats and experimental setups equipped with LED lighting. This allowed for a comparison of illuminance patterns throughout the day. Measurements were taken using a pendant temperature/light data logger (HOBO, model UA-002-64, USA). The experiment was conducted under a constant room temperature of 25 ± 2 °C.

Growth performance of W. globosa                

Following the determination of natural PPFD and illuminance values, these parameters were replicated using LED lighting for cultivation experiments. A 12:12 h (light:dark) photoperiod was applied to mimic a tropical daytime period and avoid stress from light overexposure. A 10 x 10 x 25 cm-glass containers were filled with 500 mL of water for cultivation. The experiment was conducted using a completely randomized design (CRD) with varying water sources and nutrient supplementation across treatment groups. Tap water using in this study met the quality standards of the Provincial Waterworks Authority, Thailand, with a pH of 6.5-8.5, less than 4.0 NTU turbidity and low nutrient levels.

 For the fertilized treatments (T3 and T4), a commercial compound fertilizer (N-P₂O5-K₂O: 15-15-15) was applied at a concentration of 0.15 g L⁻¹. This dosage supplied approximately 22.5 mg L⁻¹ of Nitrogen (N), 9.8 mg L⁻¹ of Phosphorus (P), and 18.7 mg L⁻¹ of Potassium (K) to the culture medium. The experiment was conducted as a single run over a 15-day period. Six experimental groups were established, each with three independent biological replicates (separate containers), resulting in a total of 18 experimental units (n = 18) (Table 1).

Table 1. Experimental groups of W. globosa cultivation under different water conditions.

A total of approximately 625 W. globosa fronds (0.1 g) were introduced into each prepared container. Growth performance was monitored every three days over a 15-day period (days 0, 3, 6, 9, 12, and 15). To estimate population density accurately, avoiding methods that cause plant biomass loss such as using dry weight or chlorophyll content (Dinh et al., 2022), a biomass-based calculation method was employed. A calibration factor was first established by weighing a random sample of 1 g of fresh W. globosa and counting the fronds using a Sedgewick-Rafter counting chamber (Menzel Gläser, Germany) under a compound microscope. During the experiment, the total wet biomass of each experimental unit was recorded, and the total population was calculated using the established mass-to-count ratio. At the end of the experiment, specific growth rate (SGR) was calculated for each 3-day interval following the method of Phatarpekar et al. (2000) and Haffner et al. (2020). The formula used was:

Where μ is the specific growth rate (day⁻¹), W_t is the estimated total frond number derived from wet biomass at day t, W₀ is the estimated total frond number at the preceding sampling day (day t-3), and t is the time interval (3 days).

Statistical analysis

Statistical analyses were conducted using both Python (Statsmodels library) and IBM SPSS Statistics version 22. To account for the repeated measurements taken on the same experimental units over time, growth data were analyzed using a Linear Mixed-Effects Model (LMM). The model included 'Treatment', 'Time', and their interaction ('Treatment × Time') as fixed effects, with 'Experimental Unit' set as a random effect to address temporal autocorrelation.

Additionally, One-way Analysis of Variance (ANOVA) followed by Duncan’s new multiple range test was used to compare treatment means at specific individual time points (e.g., final harvest). Differences were considered statistically significant at P < 0.05.

RESULTS

Light factors affecting W. globosa growth

Clear differences in PPFD were observed between natural and LED lighting. Recalculation of spectral data confirmed that the total Photosynthetic Photon Flux Density (PPFD, 400–700 nm) was 43.81 ± 8.81 µmol m⁻² s⁻¹ for natural shaded light and 44.93 ± 2.41 µmol m⁻² s⁻¹ for the LED treatment. The LED lighting displayed peak emissions at 454 nm (blue) and 655 nm (red) (Figure 1).

Figure 1. Spectral distribution of Photosynthetic Photon Flux Density (PPFD) measured in the natural shaded habitat of W. globosa (Orange line) compared to the bio-inspired LED laboratory setup (Blue line).

Illuminance measurements over a 12-hour daylight period showed that natural light ranged from 22.18 to 2,257.53 lux, with a mean of 1,187.32 ± 590.52 lux. In contrast, LED light ranged from 2,147.75 to 2,239.46 lux, with a mean of 2,192.37 ± 25.60 lux. This reflects the high variability of natural light due to environmental factors such as weather, cloud cover, and shading, with peak illuminance recorded at 12:00 reaching 2,331.92 lux. In comparison, LED lighting provided a stable and consistent illuminance throughout the experimental period (Figure 2).

Figure 2. Light illuminance during daylight hours comparing natural outdoor environments and laboratory experimental areas with LED lighting.

Growth performance of W. globosa

The Linear Mixed-Effects Model analysis confirmed that nutrient supplementation significantly altered the growth trajectory of W. globosa over time. A significant Treatment × Time interaction was observed (F_6,52 = 16.23, P < 0.001, R²_m= 0.78). This interaction was primarily driven by treatments T3 and T4, which exhibited significantly higher and sustained biomass accumulation compared to the control throughout the 15-day period.

Significant differences in specific growth rate (SGR) were observed among all treatment groups (P < 0.05). On day 3, the T2 group exhibited the highest initial SGR (0.343 ± 0.00 day^-1), followed by the T4 group (0.229 ± 0.10 day^-1). However, the growth rate of T2 declined sharply shortly thereafter. In contrast, the fertilized treatments maintained positive growth throughout the experiment. The T4 group maintained a stable rate ending at 0.083 ± 0.03 day^-1 on day 15, while the T3 group showed moderate initial growth (0.087 ± 0.04 day^-1) that improved by day 15 (0.139 ± 0.12 day^-1). The T5 group exhibited moderate initial growth (0.136 ± 0.11 day^-1) but failed to sustain this rate, dropping to -0.013 ± 0.04 day^-1 by day 15. Conversely, groups without nutrient supplementation (C and T1) exhibited decreasing growth after day 9, with negative SGR values recorded on day 12 (-0.155 ± 0.17 day⁻¹ for C and -0.394 ± 0.07 day⁻¹ for T1) (Table 2)

Table 2. Specific growth rates (SGR) of W. globosa under different cultivation conditions over 15 days.

Note: *Different superscript letters indicate significant differences among treatments on the same day (P < 0.05).

Plant counts taken every three days over the 15-day period supported the SGR findings. The T4 group produced significantly higher W. globosa counts (P < 0.05), followed by T3. In contrast, the non-fertilized groups (C, T1, and T2) showed notably lower plant numbers (Figure 3).

Figure 3. Number of W. globosa fronds under various cultivation conditions over 15 days. The red line is initial number of W. globosa fronds.

DISCUSSION

This investigation elucidates the critical interplay between light and nutrients for W. globosa development in closed cultivation systems. A key finding is that replicating the plant's natural, low-light habitat conditions can support productivity when nutrient availability is optimized. Our spectroradiometric analysis confirmed that the total PPFD in the natural shaded habitat was approximately 43.81 µmol m⁻² s⁻¹, which is significantly lower than the high intensities (100–600 µmol m⁻² s⁻¹) typically recommended for commercial duckweed cultivation (Landolt and Kandeler, 1987; Appenroth et al., 2018). Similar to light illuminance, Ketkaew and Rakthai (2022) suggested that light illuminance greater than 5,000 lux is necessary, and Chantiratikul et al. (2010) found that a light illuminance of 12,000-15,000 lux is suitable for the growth of duckweed. However, in our study, the light illuminance did not exceed 2,500 lux. Our bio-inspired LED system was calibrated to match this low-energy level (44.93 µmol m⁻² s⁻¹), but optimized with peak emissions at 454 nm (blue) and 655 nm (red) to align with chlorophyll absorption maxima. Although natural light in shaded habitats is often dominated by green wavelengths which are less efficiently used by chlorophyll compared to red and blue (Nguyen and Sung, 2024). The ability of W. globosa to thrive under such conditions suggests a strong adaptation to low-light environments. This contrasts with many cultivation studies that aim to maximize biomass primarily by applying high light intensities (Smith et al., 2024).

Our bio-inspired LED system, calibrated to mimic these low-intensity conditions, proved highly effective. The LED source provided peak emissions at 476 nm (blue) and 630 nm (red), aligning with the absorption maxima of chlorophylls and supporting the fundamental enzymatic processes of chlorophyll biosynthesis essential for efficient photosynthesis (Bian et al., 2015; Zhang et al., 2019; Kamolrat et al., 2023; Soufi et al., 2023). Crucially, the LED system delivered this optimal spectrum with high temporal stability, eliminating the diurnal and environmental variability inherent in natural sunlight. This stability is a critical advantage of closed-system cultivation, allowing for consistent and predictable growth conditions.

The interplay between light and nutrients was clearly demonstrated by the distinct growth patterns observed. Interestingly, the T2 group (tap water) exhibited the highest initial growth surge on day 3. This phenomenon was likely driven by the rapid utilization of internal nutrient reserves stored within the fronds, stimulated by the optimal light spectrum. However, this growth was short-lived; the subsequent population collapse (bloom-and-bust) in T2, as well as in other unsupplemented groups (C, T1, and T5), indicates that internal reserves and trace minerals in water are insufficient for sustained biomass production. These groups showed a rapid decline after day 9, leading to negative SGRs as internal nutrient reserves were depleted. The rapid population decline observed in these unsupplemented treatments implies a severe nutrient limitation rather than environmental stress. Although specific hydro-chemical parameters (pH, EC) were not continuously monitored, W. globosa is widely recognized as a eurytopic species with a broad tolerance to pH (4.5–10.5) and temperature fluctuations (Landolt, 1986). Therefore, the limiting factor was evidently the insufficient concentration of Nitrogen and Phosphorus, falling below the critical threshold required to sustain biomass. In contrast, the fertilized treatments (T3 and T4) maintained positive specific growth rates throughout the 15-day period. The T4 group, in particular, demonstrated that when the ‘bio-inspired’ light signals are coupled with adequate N-P-K availability (approx. 22.5 mg L⁻¹ N, 9.8 mg L⁻¹ P, and 18.7 mg L⁻¹ K), the plant can sustain continuous cell division. This confirms that under species-appropriate lighting, nutrient availability rather than light intensity is the primary limiting factor for W. globosa.

The superior and stable performance of the T4 treatment (LED + fertilized tap water) suggests a highly efficient synergistic mechanism. The blue and red wavelengths stimulate photosynthetic activity (Li et al., 2021; Liang et al., 2022; Chen and Liu, 2024). The initial dose of fertilizer created a nutrient-rich environment that allowed the plant to fully capitalize on this light energy for continuous cell division. This synergy resulted in the most consistent and effective growth, proving that readily available tap water fortified with a standard fertilizer is a more reliable medium than unsupplemented natural pond water for cultivation in a closed system.

Our conclusion that nutrient availability was the primary limiting factor is consistent with findings in related duckweed species, where nutrient concentrations were shown to be the ultimate determinant of biomass yield (Dou et al., 2017; Liang et al., 2022). Furthermore, the success of our T4 treatment aligns with the known ecological strategy of many Lemnaceae species, which are adapted to thrive in shaded, yet nutrient-rich, waters (Petersen et al., 2022). While other studies have focused on maximizing yield through high-intensity lighting, our findings present a viable, energy-efficient alternative. We demonstrate that a low-energy LED protocol (~45 µmol m⁻² s⁻¹) is sufficient to support consistent growth (SGR ~0.23 day⁻¹) when nutrient requirements are met. This approach offers a practical model for reducing operational costs in closed-system cultivation.

From a practical standpoint, our findings offer a clear and simplified protocol for W. globosa cultivation. This study provides a proof-of-concept that growers can achieve consistent yields using tap water and a basic N-P-K fertilizer, bypassing the complexities and contamination risks associated with natural pond water (Lam et al., 2018). This simplified, technology-driven approach offers a promising model for the reliable production of Wolffia as a sustainable food source to help supply the growing global protein market (Appenroth et al., 2018; Sońta et al., 2020; Xu et al., 2021).

Nonetheless, the limitations of this study should be acknowledged. The experiments were conducted on a small laboratory scale (500 mL containers) over a 15-day period. Importantly, the conclusion regarding the sufficiency of low-energy LEDs is specific to these controlled conditions and the short timeframe tested. Since biomass yield per unit area was not directly compared against a conventional high-intensity control, long-term productivity remains to be validated. Therefore, claims of commercial scalability require further investigation in larger, pilot-scale cultivation systems where factors like self-shading, gas exchange, long-term nutrient dynamics, and the need for precise thermal regulation to maintain optimal temperatures (Wedge and Burris, 1982) become more pronounced. Additionally, while the nutrient input from fertilizer was calculated, future studies should include a comprehensive analysis of the baseline water chemistry (N, P, K) of the source water to refine the nutrient supplementation strategy further. Future work should also explore varying light intensities above our bio-inspired baseline to identify an optimal point between energy consumption and biomass yield. Furthermore, an experimental comparison with outdoor cultivation under local conditions, as well as an analysis of the nutritional composition of the harvested biomass, would provide a more comprehensive evaluation of this protocol's feasibility.

CONCLUSION

This study successfully demonstrates that a bio-inspired, low-energy lighting strategy, which mimics the natural shaded habitats of W. globosa, is a viable approach for closed-system cultivation. The critical role of nutrient availability was confirmed; a one-time supplementation of fertilizer was sufficient to support sustained growth, while unenriched systems failed. Our findings establish a practical proof-of-concept that sustained productivity can be achieved without the high energy costs of high-intensity lighting. Instead, a simplified protocol combining species-appropriate lighting
(~45 µmol m⁻² s⁻¹) with basic nutrient enrichment offers a promising and efficient model for consistent Wolffia production. However, the limitations of this study regarding its short duration (15 days) and small laboratory scale must be acknowledged. Future research should focus on validating these results in larger, long-term pilot systems and investigating detailed nutrient dynamics to fully assess its potential for the sustainable protein market.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to the entire research team for their unwavering support and invaluable contributions throughout this study. Your dedication and expertise have been instrumental in the successful completion of this research. Special thanks to the collaborators for their insightful feedback and guidance, which have greatly enhanced the quality of this work. I am also grateful to Kasetsart University for providing the necessary resources and support, as well as the administrative staff for their assistance throughout the research process. Thank you all for your essential contributions to this project. Finally, I would like to thank you The Kasetsart University Research and Development Institute (KURDI), Kasetsart University, Bangkok, Thailand provided English-editing assistance.

AUTHOR CONTRIBUTIONS

Pichasit Sangmek: Data Curation (Equal), Formal Analysis (Equal), Investigation (Supporting), Methodology (Equal), Validation (Equal), Writing – Original Draft (Lead) and Writing – Review and Editing (Equal); Natakorn Thasnas: Funding Acquisition (Supporting), Investigation (Supporting), Resources (Lead)  and Software (Supporting); Suphasit Sitthaphanit: Conceptualization (Supporting), Investigation (Supporting), Supervision (Supporting), Visualization (Supporting) and Writing – Review and Editing (Supporting); Narong Kamolrat: Conceptualization (Lead), Funding Acquisition (Lead), Data Curation (Equal), Formal Analysis (Equal), Project Administration (Lead), Investigation (Lead), Methodology (Equal), Resources (Supporting), Supervision (Lead), Validation (Equal), Visualization (Lead)  and Writing – Review and Editing (Equal). All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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