Introduction

Duckweed is a small, floating aquatic plant that plays an important role in experimental-scale wastewater treatment. Due to its high biomass production, reasonable protein content, and strong anti-pollution capabilities, it has attracted increasing interest for potential use in animal feed systems and water restoration. In 2016, a report published by the Food and Agriculture Organization (FAO) indicated that 70% of the world’s freshwater extractions are for agriculture and livestock, and the pollution from manure wastewater and its adverse effects on the environment are becoming increasingly prominent1,2. Consequently, the growing demand for animal products and the emerging competition for water resources threaten the sustainability and productivity of the agricultural sector, leading researchers to investigate new techniques to optimize water use within livestock systems, such as the use of Spathiphyllum and microalgae3,4,5. Furthermore, in China, the protein source for animal feed is increasingly dependent on imports, and finding a feasible alternative is a significant challenge6. Therefore, the reuse of farm effluent for growing animal feed presents an opportunity, and the nutrient cycle model of duckweed — feedstuff — livestock offers a new strategy to reduce environmental pollution.

Duckweed is the smallest flowering plant in the Araceae family and is dispersed worldwide on the surface of freshwater. It has great potential for wastewater remediation and is considered a promising source of animal feed protein in the future. It is the most rapidly cloned angiosperm known, exhibiting exponential growth, and is the closest plant to the “Darwin-Wallace Demons”7. Under optimal conditions, its doubling time is reported to be 1.34 to 4.54 days8. It has been estimated that duckweed populations produce more than five times as much biomass per hectare per year as Zea mays, with protein content ranging from 15 to 45%, and it has significant output advantages and considerable economic importance9,10.

Some studies have shown that various types of duckweed can effectively reduce the content of pollutants (nitrogen and phosphorus) when cultivated on nutrient-rich substrates, and rapidly accumulate a significant amount of protein-rich biomass within a short time frame. Chen et al. reported that the mixed cultivation of three different species of duckweed removed 99.1% of total nitrogen (TN) and 90.8% of total phosphorus (TP) from wastewater11. In an earlier study, promising results were obtained in treating industrial wastewater using duckweed, as evidenced by removal rates of 73–84% for chemical oxygen demand (COD), 83–87% for TN, and 70–85% for TP12. According to Pena et al., duckweed removed 58.9% of COD, 66% of phosphorus, and 74% of NH3-N from pig wastewater, and recorded its highest growth rate of 3.1 g/m2/d13. Recycling pollutants from wastewater for animal feed production presents a promising opportunity for sustainable agriculture.

Recently, the application of duckweed in wastewater treatment has gained traction due to its efficient enrichment and conversion of nitrogen and phosphorus in wastewater into high-quality biomass raw materials. For example, the phyto-Fenton process of duckweed was used to efficiently remove sulfamethoxazole from contaminated wastewater, and duckweed biomass was applied in the anaerobic digestion process, which generated CH414. Fluorine can affect the aminolevulinic acid dehydrogenase and chlorophyllase activity of duckweed in chicken manure extracts, and high concentrations of fluorine can lower the growth rate of duckweed15. The addition of acetic acid to tannery and textile industry wastewater can significantly enhance the antioxidant defense mechanisms of duckweed and reduce the toxicity of heavy metals to the plant16. These researches indicate that the application of duckweed in wastewater treatment is progressing towards high efficiency, cost-effectiveness, and resource utilization.

Biogas slurry is widely known for its high concentration of organic pollutants, which exhibit a much higher pollution load compared to domestic sewage. Therefore, reducing the negative impact of biogas slurry on the environment has become one of the key challenges that urgently need to be addressed in the agricultural sector. In some developing countries, using biogas slurry as organic fertilizer has become a widely adopted practice, which helps promote the development of agriculture and animal husbandry production sectors towards a more sustainable direction17. However, biogas slurry manufacturing plants that lack sufficient agricultural land may be compelled to discharge excess wastewater to other regions. In this context, in addition to concentrating the slurry through solid-liquid separation, the development of biological treatment technologies for these slurries has become a primary focus of research efforts18.

To address these open questions, a study was conducted in an outdoor greenhouse and cultivated with piggery slurry effluents. The production characterization of the biogas slurry water and the duckweed was monitored over three weeks to determine optimal growth conditions in biogas slurry effluents. Sufficient evidence was gathered to understand the changing order of nutrients in the biogas slurry. The purpose of this study was to identify the production performance of duckweed at the optimal concentration and evaluate its protein production potential. The second purpose was to clarify the purification effects of duckweed on swine manure and biogas slurry, and to evaluate its potential application in agricultural waste treatment and ecological agriculture development. This research provides data on duckweed growth and nutrient removal at various biogas slurry concentrations, offering valuable information for the design and operation of pilot-scale and full-scale duckweed cultivation systems in the future.

Results

Identification of duckweed species

In the asp gene comparison results (Appendix B), the duckweed collected matched 99% with the genome of Lemna aequinoctialis (Sequence ID: KJ630511.1), comprising 686 bases, of which 683 were identical. In the rps gene comparison, the genome matching rate was also 99% (Sequence ID: KJ503283.1), comprising 975 bases, of which 973 were identical. Additionally, morphological references were made. The leaf-like structures of indigenous duckweed, known as fronds, are approximately 3–4 mm, oval, and possess two pouch-like structures in the proximal region, consistent with the physiological characteristics of Lemna aequinoctialis19.

Harvesting of duckweed biomass

According to the results in Fig. 1, the biomass of duckweed in the controls (CK) and 2-8% treatment groups increased over time, whereas the biomass in the 10% treatment group steadily declined. During the same time period, the biomass of duckweed in the treatment groups with increasing concentrations showed a trend of initial increase followed by a decrease. For example, on the 7th day, the 4% treatment group showed the highest accumulation of duckweed biomass, with a fresh weight of 48.3887 g. The dry weight after dehydration was 3.3477 g, representing a significant increase of 42.35% relative to the control group. Relative to CK, the dry weight in the 2% treatment group increased by 35.61%, while the 8%, and 10% treatment groups decreased by 34.56%, and 70.07%, respectively. On the 14th day, the 4% treatment group continued to maintain the highest biomass, with a fresh weight of 117.7113 g. The dry weight was 5.6866 g, representing a significant increase of 29.41% relative to CK’s 4.3941 g. The 2% and 6% treatment groups increased by 6.31% and 24.32%, respectively, relative to CK, while the 8% and 10% treatment groups decreased by 39.19% and 90.29%. On the 21st day, the biomass in the 6% treatment group surpassed all other groups, with a maximum dry weight of 9.1153 g, which was 80.80% higher than that of CK (5.0416 g). Relative to CK, the 2%, 4%, and 8% treatment groups showed increases of 64.28%, 67.01%, and 4.26%, respectively, while the 10% treatment group showed a decrease of 95.80%.

Fig. 1
figure 1

Biomass of duckweed recorded at different time points during the experiment. The upper part shows fresh weight, and the lower part shows dry weight. The data are presented as the mean ± standard error of three replicates. CK: No biogas slurry added; 2-10%: Biogas slurry added at the percentages indicated on the labels. Different lowercase letters in the figure indicate significant differences at the 0.05 level among the means at the same time point; no comparisons are made between different time points.

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The production capability of the plants was calculated based on their weight, and in Fig. 2, the production capability curve exhibited a peak-like shape. At the concentration of 4%, it reached the peak of production capacity, and then with the growth trend decreased followed by the increase of biogas slurry concentration. In other words, the production capability of duckweed cultivated in biogas slurry showed a trend of initially increasing and then decreasing. On the 7th day (Fig. 2A), the 4% treatment reached the peak, with production capability at 1.7248 g/(d·m2), while the lowest production capability was observed in the 10% treatment. On the 14th day (Fig. 2B), the curve maintained a similar pattern, but the maximum production capability decreased to 1.5585 g/(d·m2), and the minimum production capability showed a negative value (-0.0070 g/(d·m2)). On the 21st day (Fig. 2C), overall production capability further declined, with the 6% treatment showing the highest value (1.7193 g/(d·m2)). Notably, the 8% and 10% treatments consistently had lower values than the CK, with the difference being more pronounced at earlier time points. Therefore, this suggests that excessively high concentrations of biogas slurry inhibit duckweed growth.

Fig. 2
figure 2

Productivity curve of duckweed at different biogas slurry concentrations. (A), (B), and (C) represent productivity at 7 days, 14 days, and 21 days, respectively. CK: No biogas slurry added; 2-10%: Biogas slurry added at the percentages indicated on the labels. The data represent three replicates for each treatment.

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Additionally, we calculated their relative growth rate (Fig. 3) and doubling time (Fig. 4). It was observed that the relative growth rate also followed a similar pattern of increasing and then decreasing, while the doubling time exhibited an opposite trend. During the first 14 days of the experiment, the relative growth rate of duckweed in the 4% treatment consistently outpaced that of all other groups. At 7 days (Fig. 3A), the highest relative growth rate was recorded in the 4% treatment at 0.2866 g/(g·d), which was 58.17% higher than the relative growth rate at 14 days (0.1812 g/(g·d)) (Fig. 3B). By day 21 (Fig. 3C), the 6% treatment demonstrated the highest relative growth rate of 0.1433 g/(g·d). Furthermore, we organized these data (Fig. 3D), which clearly demonstrated that the longer the duckweed were grown, the smaller their relative growth rate became. At 7 days, the 4% treatment was significantly higher than the other treatment groups. After 7 days, the 10% treatment showed negative growth, and the extent of negative growth gradually increased. Doubling time analysis was conducted during the first week of most rapid duckweed growth (Fig. 4). The 4% treatment group had the shortest doubling time, of only 2.42 days. Compared to the control (CK), the doubling time was significantly decreased by 15.65% and 17.69% in the 2% and 4% treatment groups, respectively, while it increased significantly by 34.94% and 272.33% in the 8% and 10% treatment groups.

Fig. 3
figure 3

Relative growth rate of duckweed at different biogas slurry concentrations. In the upper left corner of the figure, (A), (B), and (C) represent relative growth rates at 7 days, 14 days, and 21 days, respectively. CK: No biogas slurry added; 2-10%: Biogas slurry added at the percentages indicated on the labels. The data represent three replicates for each treatment. (D) is the combined data from (A), (B), and (C), presented as the mean ± standard error of three replicates. Different lowercase letters on the bars indicate significant differences at the 0.05 level among the means at the same time point; no comparisons are made between different time points.

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Fig. 4
figure 4

Biomass doubling time of duckweed under different treatments during the first 7 days. CK: No biogas slurry added; 2–10%: Biogas slurry added at the percentages indicated on the labels. The data are presented as the mean ± standard error of three replicates. Different lowercase letters indicate significant differences at the 0.05 level among the means.

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Alteration in chlorophyll

Chlorophyll content serves as an indicator of plant growth status20. By measuring the chlorophyll content of duckweed under different conditions and at different times, it is possible to evaluate the optimal growth state and the best harvest time for duckweed. As shown in Table 1, the chlorophyll content of duckweed within the same group followed a pattern of initially increasing and then decreasing, with the passage of time, the chlorophyll content exhibited a continuous decline. The maximum chlorophyll content of 0.84 mg/g exceeded that of CK significantly and was observed in the 4% treatment group on the 7th day. The chlorophyll content of duckweed in the 2%, 4%, and 6% treatment groups was significantly higher than that of CK, whereas the chlorophyll content in the 8% and 10% treatment groups was significantly lower than that of the other treatment groups and the control. On the 7th day, there was no significant difference in chlorophyll a content among the 2%, 4%, and 6% treatment groups (Table 2). However, as the cultivation time increased, the differences in chlorophyll a content became increasingly pronounced. On the 14th and 21st days, the difference between the 4% and 6% treatment groups was not significant, but the chlorophyll a content in the 6% treatment group was significantly higher than that in the 2% group and the control. The highest chlorophyll b content was observed in the 4% treatment group on the 7th day (Table 3), but this trend did not persist, as the 6% treatment group surpassed it on the 14th and 21st days, although the difference was not significant.

Table 1 Total chlorophyll content of duckweed at different times in different treatments.
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Table 2 Chlorophyll a content of duckweed at different times in different treatments.
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Table 3 Chlorophyll b content of duckweed at different times in different treatments.
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Nitrogen and protein content

Duckweed from different treatments exhibited variations in protein content (Fig. 5). As the biogas slurry percentage increased, the protein content first rose and then declined, with the control consistently showing the lowest values. Since the protein content is directly proportional to the nitrogen content, their trends were observed to be consistent. In all collected samples, the protein content of duckweed in the 4%, 6%, and 8% treatments was above 30%, while the control (CK) consistently remained below 10%. On the 7th day, the 6% treatment showed the highest protein content (36.07%). The difference between the 6% treatment and the 2% and 4% treatments was not significant, but it was significantly higher than the 8% treatment (32.83%). After 14 days, the protein content of duckweed treated with 8% slurry reached 36.34%, marking a 3.51% increase compared to the previous measurement. The protein content of the 4% and 6% treatments decreased by 2.27% and 2.23%, respectively, while the protein content of the 2% treatment significantly dropped to 19.82%. The 21st day maintained the ranking structure observed from the previous week, with the 8% treatment continuing to remain high (34.65%). However, compared to previous measurements, the nitrogen and protein content in each group showed a downward trend.

Fig. 5
figure 5

Nitrogen content and protein content in harvested duckweed samples. CK: No biogas slurry added; 2-10%: Biogas slurry added at the percentages indicated on the labels. The data are presented as the mean ± standard error of three replicates. Different lowercase letters indicate significant differences at the 0.05 level among the data at the same time point.

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Nutrient recovery from biogas slurry

As shown in Fig. 6A, the COD concentration in the water decreased over time across all treatment groups as the number of experimental days increased. During the first 7 days of the experiment, there was a significant difference in COD removal rates among the groups (Table 4). The COD concentration in the control and 2–10% treatment groups decreased by 17.87%, 41.76%, 46.73%, 62.41%, 65.83%, and 69.91%, respectively, with higher sludge concentrations leading to greater removal rates. However, during the second and third weeks, the overall rate of COD reduction slowed, with the maximum reduction being only 9.35%, and the values across the treatment groups gradually converged. At the end of the experiment, the COD removal rates from highest to lowest were in the order of 10% > 8% > 6% > 4% > 2% > CK. The COD content in all samples was below 100 mg/L, which is the standard for treated wastewater discharge. The experimental results indicate that duckweed can reduce the COD content in sludge water, and the higher the sludge concentration, the better the COD removal effect.

Table 4 Effect of cultivating duckweed on COD and total nitrogen removal in biogas slurry water.
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As shown in Fig. 6C, the NH3-N concentration in all treatment groups exhibited a decreasing trend as the experimental duration increased. Throughout the experiment, the ammonia nitrogen (NH3-N) removal rates in all treatment groups were consistently above 93.06%. After 7 days of the experiment, all treatment groups demonstrated effective removal of NH₃-N, with removal rates ranging from 56.78 to 71.23% (Table 5). The control (CK) group had a significantly higher removal rate than the other biogas slurry treatment groups, whose removal rates decreased as the biogas slurry concentration increased. Following this period, the concentration of CK stabilized, while the other treatments continued to decrease. By the end of the experiment, the NH3-N removal rates from highest to lowest were 4% (97.25%) > 6% (97.16%) > 2% (96.22%) > 8% (93.70%) > 10% (93.06%) > CK (79.47%). There was no significant difference among the first three treatments, but they were significantly higher compared to the 8% treatment. These results suggest that duckweed can efficiently remove NH3-N at concentrations of 2%, 4%, and 6% in biogas slurry, which may represent the optimal growth conditions for duckweed.

Similarly, for TN removal, the concentration in each treatment group gradually decreased as the number of experimental days increased (Fig. 6B). After 7 days, the TN concentration in each treatment group decreased, with CK achieving the highest removal rate (85.50%), which was significantly higher compared to the other treatments (Table 4). By day 14, the concentration further decreased, indicating that nitrogen compounds in the water were primarily removed through duckweed absorption in each treatment. At the end of the experiment, the removal rates in descending order were CK (98.13%) > 2% (96.61%) > 4% (86.89%) > 6% (83.90%) > 8% (82.22%) > 10% (80.24%), corresponding to reductions of 5.77 mg/L, 17.95 mg/L, 30.88 mg/L, 44.28 mg/L, 58.47 mg/L, and 70.29 mg/L from the initial values. The results demonstrate that cultivating duckweed in biogas slurry can effectively remove TN.

Fig. 6
figure 6

Pollutant concentrations in water with different biogas slurry concentrations; (A) represents COD content, (B) represents total nitrogen content, (C) represents ammonia nitrogen content, and D represents total phosphorus content. CK: No biogas slurry added; 2-10%: Biogas slurry added at the percentages indicated on the labels. The data are presented as the mean ± standard error of three replicates. Different lowercase letters indicate significant differences at the 0.05 level among the data at the same time point.

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As shown in Fig. 6D, the concentration of TP gradually decreased over time, with the 2% and 4% treatments demonstrating superior removal rates that were significantly higher than those of the other treatments. During the first 7 days of the experiment, the 2% treatment achieved the highest removal rate, eliminating 33.81 mg/L of TP, a value significantly higher than those of the other treatments (Table 5). In the second week, the 8% treatment exhibited the highest removal rate, showing a significant difference compared to the other treatments. At the end of the experiment, the TP removal rates in descending order were: 2% (85.54%) > 4% (85.22%) > 6% (74.27%) > 8% (68.49%) > 10% (59.97%) > CK (56.25%), corresponding to reductions from the initial values of 1.15 mg/L, 1.90 mg/L, 2.56 mg/L, 3.47 mg/L, 3.80 mg/L, and 0.06 mg/L, respectively. These results demonstrate that cultivating duckweed in biogas slurry can effectively remove TP.

Table 5 Effect of cultivating duckweed on total phosphorus and ammonia nitrogen removal in biogas slurry water.
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A correlation analysis was conducted on all factors (Fig. 7). In line with our expectations, biogas slurry concentration was significantly negatively correlated with chlorophyll b, and extremely significantly negatively correlated with dry weight, productivity, and relative growth rate, while significantly positively correlated with doubling time. In other words, when the biogas slurry concentration exceeds a certain range, further increases in concentration lead to poorer duckweed growth performance, likely attributable to stress induced by the elevated concentration. Chlorophyll has a highly significant positive correlation with dry weight, productivity, relative growth rate, nitrogen content, and protein content, and a highly significant negative correlation with doubling time. It is evident that the higher the chlorophyll content in the plants, the better the growth performance and the shorter the doubling time required.

Fig. 7
figure 7

Correlation between different factors. Purple indicates a positive correlation, green indicates a negative correlation, and the deeper the color and the smaller the ellipse area, the stronger the correlation. *, ** indicate significant differences at the 0.05 and 0.01 levels, respectively.

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Discussion

Duckweed is considered a highly valuable species for phytoremediation and a novel source of edible protein21. However, its optimal growth conditions and tolerance in the context of biogas slurry wastewater cultivation require further investigation. In this study, we used pig farm biogas slurry to evaluate the growth potential of duckweed at different concentrations and its purification effect on biogas slurry wastewater. We conducted a comprehensive comparison of the growth and physiological indicators of duckweed across treatment groups, as well as the pollutant indicators in the biogas slurry. The removal of pollutants from the biogas slurry was accompanied by changes in the growth capacity of duckweed, with some concentrations directly stimulating growth while others inhibit it.

Polymerase Chain Reaction (PCR) is a useful technique that can quickly and accurately identify plant species by amplifying specific DNA fragments and sequencing them22. In our results, the match rate between the two fragment sequences and Lemna aequinoctialis was 99%, the highest among all paired varieties. Thus, we speculate that this indigenous duckweed is most likely Lemna aequinoctialis. However, our current work cannot conclusively determine complex germplasm, as no single method can clearly classify a germplasm as a distinct species. Consistent results from multiple methods are needed to enhance classification certainty, which relies not only on genetic information but also on multiple dimensions such as ecology, behavior, culture, and epigenetics23.

Plant productivity is a complex system influenced by multiple factors, such as temperature, nutrients, light, carbon dioxide concentration, inoculation amount, and interspecies relationships. In this experiment, the only variable was the concentration of the biogas slurry, while all other factors remained constant. Among them, nutrient concentration is an important variable. It has been reported that the higher the nutrient concentration in the substrate, the greater the productivity of the plant, and that productivity beyond the maximum value becomes smaller with increasing nutrient concentration24,25. The results of the present study support this statement as the peaked curves observed between digestate concentration and productivity (Fig. 2) and between digestate concentration and relative growth rate (Fig. 3) showed similar trends. The maximum productivity of Floating Pond was 1.78 g/(d-m2) under 4% treatment condition. In contrast, a huge difference was observed in the study of Devlamynck et al. which showed a maximum productivity of 9.7 g/(d-m2)26. However, in contrast to the experimental methodology reported in this study, the authors used a 0.99 m2 Petri dish, and an initial inoculum of 500 g. The relative weekly productivity was similar to the estimation of our results.

Relative growth rate (RGR) of various puffballs has been described in many reports which show a wide variation in growth potential. The reported RGR of different puffball species ranges from 0.13 to 0.78 g/(g-d)27. Even the RGR of the same species varies considerably, with Lemna gibba showing RGRs of 0.15, 0.2855, and 0.426, respectively28,29.In the present study, the maximum RGR of Lemna aequinoctialis was in the 4% treatment, which showed a RGR of 0.29 g/(g-d), which is much less than 0.56 g/(g·d)30 reported by Datko et al. However, our results are very close to the maximum RGR of Spirodela polyrhiza (0.3 g/(g·d))31. The available data indicate that both inter- and intra-species RGR showed variability, suggesting that the RGR of puffballs is not a species or genus-specific growth attributes are reliable indicators27. Rather, specific RGR may only reflect the potential for cloning under a particular cultivation model, which is consistent with the findings of Bergmann et al.32.

Chlorophyll, as the primary pigment in plant photosynthesis, directly influences the photosynthetic rate33. No morphological changes were observed in any of the analyzed plant samples. In the first week, chlorophyll content peaked in the 4% and 6% treatment groups, indicating that duckweed grew most vigorously in biogas slurry concentrations of 4% and 6%. However, as the concentration of biogas slurry increased, we observed that the duckweed leaves tended to turn yellow, and the chlorophyll content also decreased. This could be attributed to the damage and growth inhibition caused by high concentrations of NH3-N on the leaves, as NH3-N affects the efficiency of photosynthesis and chlorophyll synthesis in plants34,35. As the cultivation time extended, a decrease in chlorophyll content was also observed in successive samples, which was consistent with the negative correlation results of correlation analysis. This can partially explain the long-term nutrient variations in the biogas slurry water, as the growth of duckweed can deplete the enriched nutrients in the water36. It is noteworthy that in all analyzed plants, the content of chlorophyll a was 1 to 2 times that of chlorophyll b. Whether in duckweed grown under optimal conditions or in stressed plants, chlorophyll a typically dominated in quantity37,38. In the first week, although the differences between the 4% and 6% treatment groups were not significant, the biomass data suggested that the duckweed in the 4% treatment exhibited superior growth.

Duckweed is a widely studied subject in research on new protein sources. In feed, a replacement rate of 10–50% with duckweed can achieve positive growth outcomes in animals such as fish fish39, poultry40, swine41, and ruminants42, indicating that duckweed has the potential to become a viable source of plant protein in animal feeds. The protein content of duckweed ranges from 15–45%43. For example, in Hafiz’s study, duckweed grown at a concentration of 30 ppm achieved a maximum protein percentage of 33%44. In our study, the highest crude protein content was 36.25%, occurring in the first week under the 6% treatment, with an average protein content of 36.07% for this group. At the same time, the protein content in the 2% and 4% treatments showed no significant difference from the 6% treatment, indicating that the protein content of duckweed cultivated in biogas slurry remains high, with 6% being the optimal accumulation concentration. However, over time, the protein content of duckweed showed a declining trend, with the 8% treatment exceeding that of other treatments and showing significant differences, while the protein content in the 2% treatment remained at around 19% after the first week. This may be related to the depletion of nutrients at low concentrations and the reduction of high concentrations to the optimal accumulation level within the system. A small amount of COD, NH3-N, TN, and TP was detected in the water samples from the 2% treatment (Fig. 6), and the protein content of duckweed varies under different growth conditions10,45,46. Although the protein content of the 8% treatment was surprisingly high, its yield was unsatisfactory, with dry matter weights 54.05%, 53.01%, and 37.58% lower than the 4% treatment across three measurements, likely due to the inhibition of biomass accumulation by excessively high NH3-H levels35.

Changes in pollutant levels in biogas slurry wastewater can more directly reflect the remediation ability of duckweed. Component analysis of the biogas slurry revealed the high efficacy of duckweed in its remediation. In this study, the COD removal rate ranged from 47 to 79%, which exceeded Adhikari’s secondary duckweed wetland model (-40–40%) and approximated the primary model (20–80%)47. The COD decreased significantly during the first week, dropping below 85 mg/L in all groups, and the differences among the groups also narrowed. Interestingly, despite the lack of growth in the 10% treatment group during these 7 days, this group had the highest removal rate. This phenomenon warrants further investigation and may be attributed to the low initial inoculation density, which was insufficient to extensively cover the water surface. Sunlight penetrated the duckweed mat, leading to rapid microbial and phytoplankton growth in the tank, thereby accelerating COD degradation48. Additionally, a moderate concentration of biogas slurry can enhance photosynthesis in the plants, promoting increased oxygen release from the duckweed roots, consequently accelerating the degradation of organic matter in the water49.

Ammonia nitrogen constitutes the primary form of nitrogen in biogas slurry wastewater, and duckweed preferentially utilizes NH3-N over NO3-N, making it an ideal candidate for removing NH3-N from aquaculture wastewater50,51. In this study, duckweed achieved NH3-N removal rates exceeding 90%, with the highest rate (97.25%) observed at a 4% dilution. Duckweed’s efficient NH3-N removal is likely attributed to its direct involvement in the glutamine synthetase/glutamate synthase enzyme system for protein synthesis52. However, duckweed tolerance to NH3-N toxicity appears limited. Rooijakkers research indicates that nitrogen concentrations exceeding 60 mg/L in aquatic environments can induce toxicity in duckweed53. Another study demonstrated that NH3-N concentrations above 50 mg/L reduced duckweed NH3-N absorption efficiency51. Our findings align with these observations, as duckweed perished in the 10% treatment group, and relative growth rate (RGR) declined in the 8% treatment group. Furthermore, elevated NH3-N concentrations can induce oxidative stress in duckweed, leading to increased superoxide dismutase activity and plasma membrane damage, which may underlie its mortality in high-concentration treatment groups54. While studies have examined the antioxidant responses of duckweed under various stress conditions, further investigation into its response to high NH3-N concentrations is essential for optimizing its role in wastewater remediation and biomass production. In this study, accurately monitoring ammonia nitrogen concentrations in the substrate is critical for effective duckweed cultivation. In practical applications, ammonia nitrogen concentration should be used as a key control parameter to determine the optimal biogas slurry dilution rate.

Duckweed primarily absorbs nitrogen through its leaves and roots. Hashimi and Joda reported that duckweed could remove 96% of TN in domestic sewage55. Another study demonstrated that Spirodela oligorriza could remove 89.4% of TN from 6% swine lagoon water45. This aligns closely with our results, where the TN removal rate for each treatment group of Lemna aequinoctialis ranged from 80.24 to 96.61%. The reduction of TN is partially attributed to absorption by duckweed’s roots and leaves, as plant uptake represents only one mechanism in duckweed wastewater treatment systems56. Beyond root absorption, the anaerobic microzones beneath duckweed can facilitate microbial denitrification. Studies indicate the presence of anaerobic microzones beneath water surfaces covered by plants57. These zones may induce microbial denitrification, particularly at night when photosynthesis is inactive, leading to oxygen depletion and promoting conditions suitable for denitrification58. Additionally, the emission of volatile nitrogen compounds (NH₃ and N₂O) also contributes to the decrease in TN concentration in the substrate. Thomas et al. reported that NH₃ emissions were 0.15 g/m2/h when pig manure biogas slurry was stored in lagoons, increasing to 0.21 g/m2/h when stored in tanks59.

In the wastewater treatment system of this study, unlike nitrogen removal, phosphorus removal primarily occurs through duckweed absorption. Previous studies have shown that duckweed can effectively absorb 90.8% of phosphorus from wastewater, storing it as endogenous phosphorus11. Another study reported a phosphorus absorption rate as high as 98.8%46. In our study, the highest phosphorus removal rate achieved was 85.54%. In addition to plant absorption, phosphorus is also removed through adsorption onto the biofilm and container walls associated with duckweed. The varying removal rates may be attributed to differences in the systems in which they operate. Pollutant sedimentation and microbial activity in complex systems can account for part of the phosphorus removal46,60. Song et al. reported that duckweed exhibited optimal growth at phosphorus concentrations of 0.5-5 mg/L61. Our findings support this conclusion, as leaf yellowing in duckweed was observed in treatment groups (8% and 10%) with phosphorus concentrations exceeding 5 mg/L, while other duckweeds grew luxuriantly. Treatment groups with high phosphorus concentrations exhibited low removal rates, potentially due to salt stress inhibiting plant growth and reducing phosphorus demand. Another possible explanation is the competitive interaction between chloride ions (Cl⁻) and phosphate ions (PO43−)62.

Duckweed demonstrates strong phytoremediation capabilities and generates valuable biomass in diverse types of wastewater. It holds significant potential for applications in aquatic environmental remediation and biomass utilization. In a report similar to ours, Lemna minor was shown to produce feed materials meeting basic quality standards in media containing 0.75–1.50% pig slurry concentrations63. Unlike our study, the pig manure utilized in their research may not have undergone anaerobic fermentation. Stadtlander et al. demonstrated that duckweed cultivated in a 1:16 diluted chicken manure matrix exhibited a production capacity similar to our results, though their protein content reached up to 42.8%64. Conversely, earlier studies indicated that duckweed could absorb nutrients from cow dung slurry (1:8) with a higher dilution ratio. Despite the total NH3-N in the matrix being only 19 mg/L, the removal rate was optimal, aligning with our findings65. Furthermore, adjusting the pH of the duckweed growth substrate or introducing additional carbon sources can enhance its adaptability to wastewater concentrations and boost biomass production66,67.

As expected, the correlation analysis revealed a significant negative correlation between biogas slurry concentration and certain growth performance indicators. Interestingly, chlorophyll was positively correlated with dry weight, productivity, and relative growth rate, and negatively correlated with doubling time. Previous studies indicate that an increase in chlorophyll content is often accompanied by improvements in productivity and relative growth rate68,69. High chlorophyll content enhances the rate and efficiency of photosynthesis, which in turn affects the accumulation of organic matter and increases dry weight in plants70. This suggests that chlorophyll content may serve as an indicator of the growth status of duckweed. The potential reasons are as follows: first, changes in chlorophyll content reflect the rate of photosynthesis71; second, chlorophyll content can serve as an indicator of environmental stress, as it decreases significantly under stress conditions72; third, changes in chlorophyll content may indicate physiological responses in plants73. Recent advancements in spectral imaging technology have enabled the monitoring of plant phenotypic information to assess growth and health status74. Our results suggest that chlorophyll content may be a key indicator of duckweed growth, making chlorophyll fluorescence imaging (CFI) a promising tool for future development. The principle behind CFI is that it captures fluorescent signals emitted by chlorophyll molecules during photosynthesis, which can then be used to assess plant growth and health status75. In the future, when applying duckweed to remove biogas slurry pollutants, spectral imaging techniques such as CFI can be used to evaluate duckweed growth status and inform optimal production management decisions, thereby enhancing the plant’s ability to purify wastewater.

In summary, cultivating duckweed in biogas slurry wastewater at concentrations of 4% yields optimal results, as the duckweed demonstrates superior dry matter production capacity, protein content, and pollutant removal efficiency compared to other concentrations. Based on the initial pollutant concentrations in the biogas slurry wastewater for each treatment group (Fig. 6), the optimal growth conditions for duckweed correspond to a chemical oxygen demand of 121.77 mg/L, total nitrogen of 35.53 mg/L, total phosphorus of 2.23 mg/L, and an ammonia nitrogen concentration of 29.56 mg/L. Under these conditions, the dry matter production capacity of duckweed is 1.78 g/(d·m2), the relative growth rate is 0.29 g/(g·d), and its crude protein content is 36.25%. For duckweed cultivation and management, chlorophyll content may be a reliable observation indicator, as it strongly correlates with dry weight, productivity, relative growth rate, and doubling time in correlation analyses.

Conclusions

This experiment investigated the effect of biogas slurry concentration on duckweed growth, specifically focusing on the growth potential of Lemna aequinoctialis under suitable slurry conditions. In this study, the optimal biogas slurry concentration for cultivating Lemna aequinoctialis was found to be 4%, corresponding to an ammonia-nitrogen concentration of 29.56 mg/L, offering a crucial reference for the practical application of duckweed in removing pollutants from swine biogas slurry. However, the optimal biogas slurry dilution rate is highly dependent on the quality and source of the biogas slurry, requiring adjustments in practical applications based on the specific ammonia-nitrogen concentration to achieve optimal utilization. The results of this study provide an important reference for the operation of the duckweed production system, which is crucial for the design and operation of pilot-scale and large-scale duckweed production systems moving forward. Future studies should consider the growth kinetics and substrate consumption of duckweed in biogas slurry to achieve higher growth rates and greater biomass accumulation, thereby enhancing its purification efficiency, economic benefits, and reducing environmental pollution.

Materials and methods

Material source

This experiment tested the optimal concentration of biogas slurry for the growth of duckweed. Samples of duckweed were collected from Dongrui Food Group Co., Ltd., Heyuan, Guangdong Province, China. To determine the species of duckweed provided, DNA was extracted from samples using a plant genomic DNA extraction kit (Tengen, DP305-02), PCR-amplified, sequenced, and compared to published sequence data on the NCBI website (https://www.ncbi.nlm.nih.gov/). These results identified the species of the collected samples. Prior to the experiment, the collected duckweed samples were propagated in Hogland nutrient solution (Appendix A) to ensure uniform clonal populations across groups.

The experiment utilized swine manure biogas slurry as a growth substrate for duckweed, collected from the Dongrui Food Group swine farm (114.84, 24.15) and stored in a 1000 L IBC container in a greenhouse. The swine farm producing the biogas slurry discharges 50 m3/d of wastewater and is equipped with a 1250 m3 fully mixed anaerobic reactor (CSTR) capable of treating 25 days of wastewater, along with a digester having a capacity of 17,136 m3. The CSTR employs medium-temperature digestion with a hydraulic retention time (HRT) of 12 days. The system’s feed flow rate is 50 m3/h, and its biogas production reaches 30,000 m3/d.

Experimental design

The experimental biogas slurry was applied as follows: CK (no biogas slurry) and treatments with 2%, 4%, 6%, 8%, and 10% biogas slurry concentrations. The dilution rate was determined based on the volume ratio (v/v), and tap water was used as the diluent (Table 6). The N concentrations were 5.88 mg/L, 18.58 mg/L, 35.53 mg/L, 52.78 mg/L, 71.12 mg/L, and 87.60 mg/L, respectively, and the P concentrations were 0.11 mg/L, 1.34 mg/L, 2.23 mg/L, 3.45 mg/L, 5.06 mg/L, and 6.34 mg/L, respectively. The slurry was poured into plastic containers (60 cm × 40 cm × 35 cm) and filtered through 300-mesh gauze to remove impurities. The slurry was left to rest and aerate for one day, after which 10.0 g of duckweed was inoculated into each group’s tanks. The experiments were run synchronously and replicated nine times for both treatments and controls. The study was conducted in the Dengta Basin (114.78, 24.10), Heyuan City, Guangdong Province, China, in a greenhouse. The experiment was conducted in May, with daytime temperatures in the greenhouse ranging from 30 to 36 ℃ and nighttime temperatures between 26 and 30 ℃, and a light exposure duration of 13.2 h.

Table 6 Application methods of duckweed growth substrate.
Full size table

Sample collection

Every seven days, destructive samplings were conducted as follows: plants from three tanks per treatment were extracted, and all duckweed in each tank was collected. Additionally, 40 mL water samples were taken from each tank and stored at -80 °C. At each collection, water was removed from the duckweed’s surface with absorbent paper, the green weight was recorded, and dry weight values were obtained for each treatment after drying in an air oven at 105℃ for 48 h. These data were used to determine the percentage of dry weight, protein content, and productivity of each experimental unit.

The growth of duckweed in different concentrations of biogas slurry was evaluated using the relative growth rate (RGR, g/(g·d)) Eq. (1)76, while the biomass accumulation capacity and growth rate were assessed using productivity (P, g/(d·m2)) Eq. (2)76 and doubling time (PDT) Eq. (3)77.

$$\:RGR=\frac{{ln\:W}_{2}-{ln}{W}_{1}}{T}$$
(1)
$$\:P=\frac{{W}_{2}-{W}_{1}}{T\times\:S}$$
(2)
$$\:\text{PDT}=\frac{\text{ln}2}{RGR}$$
(3)

Here, W1 and W2 represent the dry weight of duckweed before cultivation in biogas slurry (g) and the dry weight of harvested duckweed (g), respectively. T is the cultivation time (d), and S is the cultivation area (m2).

Sample measurement and data analysis

The pigment content of the chloroplast was determined as follows: 0.2 g of fresh duckweed leaf was weighed, 95% ethanol was added and fully ground, then filtered through filter paper, and absorbance was measured with a spectrophotometer78. Generally, the maximum absorption peaks of chlorophyll a and b in 95% ethanol are at 665 nm and 649 nm, respectively, and that of carotenoids is at 470 nm79. Thus, the following equations are used to calculate the concentration of chlorophyll:

$$\:{\text{C}}_{\text{a}}\text{=13.95}{\text{\:A}}_{\text{665}}\text{-6.88\:}{\text{A}}_{\text{649}}$$
(4)
$$\:{\text{C}}_{\text{b}}\text{=24.96\:}{\text{A}}_{\text{649}}\text{-7.32\:}{\text{A}}_{\text{665}}$$
(5)
$$\:{\text{C}}_{\text{x,c}}\text{=}\frac{\text{1000}{\text{A}}_{\text{470}}\text{-2.05\:}{\text{C}}_{\text{a}}\text{-114.8\:}{\text{C}}_{\text{b}}}{\text{245}}$$
(6)

where Ca and Cb are the concentrations of chlorophyll a and b, respectively; Cx, c is the total concentration of carotenoids; A665, A649, and A470 are the absorbances of chloroplast pigment extracts at 665 nm, 649 nm, and 470 nm, respectively. Adding Eqs. (4)80 and (5)80 provides the total chlorophyll concentration (6)80.

After calculating the pigment concentration, the content of each pigment in duckweed can be obtained using the following model, which can be expressed in mg/g fresh weight or dry weight:

$${\text{Chlorophyll}}~{\text{content}}\left( {{\text{mg/g}}} \right){\text{ = }}\frac{{{\text{C}} \cdot {\text{V}} \cdot {\text{N}}}}{{{\text{m}} \cdot {\text{1000}}}}$$
(7)

in Eq. (7)80, C is the pigment content (mg/L), V is the volume of the extract (mL), N is the dilution factor, m is the sample mass (g), and 1000 represents 1 L = 1000 mL. When significant differences were detected, Fisher’s least significant difference (LSD) test was used to separate means, with a significance level of p < 0.05.

To determine the protein content in duckweed, TN was analyzed using a water-exemption condensation nitrogen analyzer (GNADA, N310) and the semi-micro Kjeldahl method81. The dried duckweed sample was ground in a mortar and sieved through a 0.5 mm sieve. Subsequently, 0.5 g of the sample was placed in a digestion tube containing concentrated sulfuric acid and hydrogen peroxide. Kjeldahl nitrogen was then measured using a digestion instrument and a scrubber. The protein content was calculated by multiplying the nitrogen content by a factor of 6.2582.

The biogas slurry was the substrate applied in the experiment, and the main pollutants included COD, NH3-N, TN, and TP. The consumption of each pollutant was measured. COD was determined by the Fast digestion-Spectrophotometric method83 and toolkit, TP by the Anti-Mo-Sb Spectrophotometric method84 and toolkit, TN and NH3-N by the UV spectrophotometry method85 and toolkit. According to the kit’s operating instructions, a multi-parameter water quality meter (Greencarey, GL900) was used to measure the concentrations of COD, NH3-N, TN, and TP in the water.

Statistical analysis

All collected data were organized and calculated, with statistical analysis conducted through one-way ANOVA in SPSS software (version 22.0, IBM SPSS Inc.). The Least Significant Difference (LSD) test was employed to determine significant differences between means, with a significance level set at p < 0.05 (data were normally distributed, with homogeneous variance). All data are expressed as means with standard errors (SE). Data visualization was performed using Sigmaplot 14.0 software. Finally, correlation analysis was conducted on all collected factors and visualized using Origin (version 2022) software.