From Waste to Resource: Use of Lemna minor L. as Unconventional Fertilizer for Lettuce (Lactuca sativa L.)

Abstract

Duckweeds, such as Lemna minor L., are invasive aquatic species that can proliferate on the surface of the nutrient solution in hydroponic systems, requiring removal operations from the cultivation tanks and disposal as waste. Several studies have demonstrated the potential use of duckweeds as an organic fertilizer. Recycling plant waste as a nutrient source for crops may be a circular approach to enhancing the sustainability of intensive horticultural production systems. Two pot experiments were carried out to evaluate the possibility of using the biomass of Lemna as a fertilizer for lettuce. The following fertilization treatments were applied: Control (no fertilization), Lemna biomass (60, 120, and 180 kg ha⁻¹ nitrogen), urea (60 kg ha⁻¹ nitrogen), and commercial organic fertilizer (60 kg ha⁻¹ nitrogen). Lettuce head diameter, fresh and dry weight, the number of leaves, and the contents of minerals, nitrates, chlorophyll and carotenoids were determined. In addition, nitrogen use efficiency was calculated. Fertilization with Lemna resulted in a significant increase in yield compared to control (+50% considering the average of the three Lemna doses) and both inorganic (+65%) and organic (+71%) fertilization treatments. No differences in yield and quality were observed between the three doses of Lemna, but the lowest one was the treatment with the best performance in terms of N productivity. These results suggest that Lemna biomass may be a proper source of nutrients for lettuce with advantages for yield and no effect on quality. Therefore, its use as an alternative to commercial fertilizers can allow farmers to profitably exploit a waste product and, at the same time, reduce the costs for fertilization, thus achieving environmental and economic benefits.

1. Introduction

Duckweeds, of the Lemnaceae family, are aquatic angiosperms characterized by small size, simple morphology, rapid growth, and a great capacity to absorb solutes from the medium in which they grow [1]. They include 36 species belonging to five genera, namely, Spirodela Schleid., Landoltia Les & Crawford, Lemna L., Wolffiella Hegelm., and Wolffia Horkel ex Schleid [2]. Duckweeds are able to adapt to almost all climate zones of the world. These plants grow on or just below the surface of water in ponds, ditches, slowly flowing streams, and other small water bodies, which they can colonize rapidly due to their extraordinary growth rate by clonal propagation [3].

The vegetative body of a duckweed plant is composed simply of a leaf-like structure called a frond, and only the genera Spirodela, Landoltia, and Lemna show roots [1]. In Lemna minor L., for example, the frond is oval to elliptical, about 6–8 mm in size, and there is a single root up to 2 cm long [4]. Daughter fronds, up to 18 per single mother plant depending on the species, originate from the meristematic region present in the proximal part of the mature frond, to which they remain connected for a time, forming colonies of 2 to 50 individuals [3,5]. Under optimal conditions, colonies can double in biomass even in just two days [6]. This behavior makes duckweeds very invasive, with ecological repercussions and economic impacts [7]. Agriculture can also be impacted, e.g., L. minor is a major aquatic weed of rice fields [8,9]. An increasing occurrence of duckweed invasion in ditches and drainage channels, driven by eutrophication and climate change, may disrupt their agricultural function and elevate management burdens [10,11]. In static hydroponic systems, such as the floating system, duckweeds find optimal conditions to proliferate on the surface of the nutrient solution not covered by the crop (Figure 1), reducing the fertilizer use efficiency due to nutrient uptake, and necessitating cleaning operations of the cultivation tanks followed by the disposal of the biomass as a waste.

On the other hand, the anatomical and physiological traits of these aquatic macrophytes and their great adaptability make them suitable plants for both basic research and numerous commercial applications [1,12]. Duckweeds can be used for the phytoremediation of diverse chemical pollutants from waters [13], the production of biofuels [14], as a pharmaceutical resource [15], as a feed ingredient in sustainable livestock production and aquaculture [16], and even for human food [17]. Furthermore, several studies report that duckweeds can be a source of nutrients for agricultural crops as a viable alternative to commercial fertilizers [18,19,20,21,22,23,24,25,26]. Fertilizers, which are essential for agricultural production and consequently for ensuring food security, are mostly produced from non-renewable raw materials, with high costs and energy consumption [27]. In addition, the use of fertilizers containing nutrients, especially nitrogen (N), in highly soluble forms raises environmental pollution concerns [28]. On the contrary, the use of bio-based fertilizers reduces the leakage of nutrients into the environment and the depletion of non-renewable resources [25,29]. The first crop in which duckweed was applied as a source of nutrients was rice [30]. Most recently, duckweed biomass was successfully used as an unconventional fertilizer in sorghum [21,23] and different vegetables, namely, tomato [18,25], Swiss chard and beet [19,20,23], spinach [20], red spinach [26], and kale [23]. Better results were obtained with duckweed green biomass than composted biomass [18]. The effect of the use of duckweed on crop yield depends on its initial elemental composition, which is affected by the effluent on which it grew [20]. Li et al. [21] and Li et al. [22] suggested the possibility of even enriching duckweed biomass with specific micronutrients, namely, zinc (Zn) and selenium (Se), to obtain fertilizers aimed at the biofortification of produce.

This study aims to evaluate the effectiveness of duckweed (L. minor) as a biofertilizer for lettuce (Lactuca sativa L.), assessing its suitability as a sustainable alternative to synthetic fertilizers. Lettuce is one of the most important leafy vegetable, whose consumption is widespread worldwide [31], and it has not been considered in studies on duckweed before. Lemna was collected from the tanks of a floating system in a farm producing vegetables both in hydroponics and in the soil, and was used in the farm itself to grow lettuce in the soil. Distributing Lemna in the soil could be a strategy to recover the carbon and nutrients accumulated in it, and to reintroduce them within the same farming system. In this way, duckweed would be transformed from waste into a resource, in a bio-based circular economy approach. To our knowledge, this is the first study that considers the possibility of exploiting the duckweed biomass obtained as a by-product of hydroponic growing systems.

2. Materials and Methods

2.1. Experimental Site and Trials Description

The study was carried out at the farm “Azienda Agricola Cammelli” (Florence, Italy; 43.781660° N, 11.158053° E), which produces vegetables both in soil and hydroponically (floating system). The study included one summer (transplanting on 14 June 2022 and harvest on 15 July 2022) and one autumn (transplanting on 10 October 2022 and harvest on 15 November 2022) growing cycle of lettuce (Lactuca sativa L.). The trials were conducted in a polyethylene-covered high tunnel, 6 m wide and 3 m high, equipped with a central suspended irrigation pipe with sprinklers spaced 1 m from each other. The temperature and relative humidity inside the tunnel during the trials were monitored using a HOBO data logger (Onset, Bourne, MA, USA). In the summer growing cycle, the maximum temperatures recorded during the day were constantly above 30 °C, while during the night they never fell below 15 °C (Figure 2a). The average daily temperature was between 25 °C and 30 °C. The relative humidity never exceeded 80% and was often below 50% for several hours during the day (Figure 2a). During the autumn growing cycle, the average minimum and maximum temperatures were 11.2 °C and 25.6 °C, respectively, with absolute values of 4.7 °C and 31.0 °C, respectively (Figure 2b). The average humidity was 84.3% (Figure 2b).

Lettuce cv. Pereion (‘Gentilina verde’ type), recommended for the summer season, and cv. Kiela (‘Lollo’ type), suitable for the autumn season, were used in the two trials, respectively. Plants were transplanted at the stage of the third–fourth true leaf in pots 20 cm in diameter (5 L in volume) filled with sandy-loam field soil collected in the tunnel and approximately 3 cm of expanded clay (at the bottom of the pot) to ensure water drainage. The main chemical properties of the used soil are reported in

Table 1. Chemical characterization of the soil used in the trials.

Parameter	Units	Value
pH		7.9
Electrical Conductivity (EC)	dS m−1	2.1
Salinity	‰	2.7
Total Nitrogen	N g kg−1	1.8
Available Phosphorus	P2O5 mg kg−1	84
Exchangable Potassium	K2O mg kg−1	345
Organic Matter	%	2.9
C/N ratio		9.6
Total Limestone	%	3.4
C.E.C.	meq 100 g−1	15.8

Analyses were carried out following the Italian official methodology indicated by the Italian Ministry of Agriculture and Forestry [32].

. The soil analysis was carried out following the Italian official methodology indicated by the Italian Ministry of Agriculture and Forestry [32].

Plants were irrigated following crop needs and considering the environmental conditions in order to avoid the risk that the water would represent a limitation factor. Three different doses of Lemna minor L. (from here on, Lemna) were distributed (L60, L120, and L180 corresponding to 60, 120, and 180 kg ha⁻¹ N, respectively), where 60 kg ha⁻¹ inorganic N is the N fertilization usually adopted by the farm for lettuce. Lemna was collected from the tanks of the floating cultivation system present in the farm and placed for 10 days on a non-woven fabric sheet inside a greenhouse for drying to a constant weight. During the drying phase, the biomass was spread in a thin layer to promote uniform drying and avoid decomposition phenomena. The greenhouse was kept constantly open to allow aeration. After the drying stage, Lemna was analyzed for elemental composition (

Table 2. Elemental characterization of Lemna used in the trials.

Parameter	Units	Value
Organic C	%	39.6
Total N	%	4.3
NO3-N	%	<0.1
NH4-N	%	<0.1
P	%	2.4
K	%	5.3
Ca	%	4.0
Mg	%	0.7
Na	%	0.3
Cl	%	1.3
S	%	1.0
Fe	ppm	13,500
Mn	ppm	350
Cu	ppm	78
Zn	ppm	97
B	ppm	573
Mo	ppm	4.0
C:N ratio		9.2

Analyses were carried out following the Kalra Reference Methods for Plant Analysis [33].

) following the Kalra Reference Methods for Plant Analysis [33]. Lemna was incorporated into the soil (3 cm depth) 28 days before lettuce transplanting in order to ensure mineralization [19]. During this period, the pots were maintained at a constant temperature of 25 °C and at field capacity, which was restored by weighing twice a week. Fertilization treatments also included a control with no fertilization (Control) and two positive controls, that is, urea (46% N) as a mineral fertilizer (Urea) and Fertorganico (11% organic N, of which 5% organic soluble N; ILSA S.p.A. Vicenza, Italy) as a commercial organic fertilizer (COF). Both Urea and COF were used at the dose normally adopted by the farm (60 kg ha⁻¹ N). Fertorganico was utilized only in the autumn trial. Urea and COF were incorporated into the soil at lettuce transplanting, according to the farm’s normal practice.

Table 3. Fertilizer treatments and respective N supply (kg ha⁻¹). COF = commercial organic fertilizer; L = Lemna.

Trial	Treatment	N Dose (kg ha−1)	Dose of Fertilizer (g pot−1)
Summer	Control	0	-
	Urea	60	0.5
	L60	60	4.7
	L120	120	9.4
	L180	180	14.1
Autumn	Control	0	-
	Urea	60	0.5
	COF	60	1.8
	L60	60	4.7
	L120	120	9.4
	L180	180	14.1

shows the N doses supplied per hectare for each fertilization treatment and the corresponding amounts of fertilizer per pot, calculated based on the fertilizer N concentration and the pot surface area. The experiment was arranged in a completely randomized block design with three blocks and three replicates (one replicate = one pot) per block per treatment.

2.2. Crop Measurements

During the growing cycle, head diameter was monitored weekly. At harvest, head fresh and dry (after oven drying at 80 °C for 48 h) weight, dry weight % (as dry weight/fresh weight × 100), and the number of leaves were determined. The leaf contents of total N, phosphorus (P), potassium (K), nitrate and, only during the autumnal trial, chlorophyll and carotenoids were analyzed. The mineral composition was determined following Kalra Reference Methods for Plant Analysis [33]. The nitrate concentration was measured using the salicyl-sulfuric acid method according to Cataldo et al. [34]. Chlorophylls and carotenoids were extracted from fresh tissues (about 50 mg) using methanol 99.9% as solvent. Samples were kept in the dark at 4 °C for 24 h, and the assays were carried out immediately after extraction. Chlorophyll a (Chl a) and chlorophyll b (Chl b) were determined by measuring the absorbance at 665.2 nm and 652.4 nm, respectively. Total chlorophyll content (Chl ab) was determined as the sum of Chl a and Chl b. Carotenoid content was computed by measuring the absorbance at 470 nm. The pigment concentrations were calculated by Lichtenthaler’s formula [35].

2.3. Nutrient Use Efficiency

For the different fertilization treatments, the nitrogen use efficiency (NUE) index was evaluated by calculating its components according to Elia and Conversa [36]: (a) partial factor productivity of the applied N (PFP), which is the amount of product harvested per unit of N applied; (b) agronomical NUE (NUEa), which measures the increase in yield obtained per unit of N applied; (c) physiological NUE (NUEp), which represents the increase in yield per unit increase in N uptake ascribable to the fertilizer (it expresses the efficiency with which the plant utilizes the N absorbed from the fertilizer); (d) apparent N recovery efficiency (REC), which represents the increase in N uptake ascribable to the fertilizer per unit of applied N (it expresses the proportion of the N applied taken up by the plants). In our study, the NUE indices were calculated on a per plant basis, and considering yield both as fresh and dry weight [37].

The used formulas were the following:

(a)

PFP = Yf/NA (g g⁻¹)

(b)

NUEa = (Yf − Yc)/NA (g g⁻¹)

(c)

NUEp = (Yf − Yc)/(TNf − TNc) (g g⁻¹)

(d)

REC = TNf − TNc/NA (g g⁻¹)

Here, Yf is the lettuce fresh/dry weight (g plant⁻¹) obtained with the N applied (NA, g plant⁻¹) with a given fertilization treatment; Yc is the lettuce fresh/dry weight (g plant⁻¹) obtained without fertilization (Control); TNf and TNc are the N contents in lettuce heads (g plant⁻¹) obtained with or without fertilization, respectively.

2.4. Statistical Analysis

Data were analyzed separately for the two trials. The statistical analysis was carried out using the CoStat 6.400 software (Co Hort, 188, Monterey, CA, USA; CoStat 2008). A one-way analysis of variance (ANOVA) was conducted and the averages were compared using the Tukey test (p ≤ 0.05).

3. Results and Discussion

3.1. Lemna Composition

Lemna is well known for its high content of macronutrients essential for plant growth, especially N and P [21,38], and micronutrients such as iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu) [39,40,41]. However, its composition can vary based on the characteristics of the medium in which it grows [42]. The Lemna used in this trial was collected from a hydroponic system where basil was grown, and thus, it floated on a nutrient solution. The N content was about 4.0% (Table 2), an amount slightly lower than that observed by Chikuvire et al. [19] and Kreider et al. [21] but compliant with the definition of organic fertilizer according to the Italian Law on fertilizers [43]. Phosphorus concentration (Table 2) was higher than that observed by different authors [19,44,45], while K was consistent with the values reported by Kreider et al. [21] and Vladimorova and Georgiyant [45]. The literature reports values highly variable for micronutrients [39,40,41,44], but within the same order of magnitude as the values we observed. Only for Fe did we find significantly higher values, probably due to the fact that the hydroponic nutrient solution contained chelated Fe, a form easily available for plants. However, the Fe content of Lemna (Table 2) was within the typical range of Fe concentration in fertilizers (0.1–5.0%). The C/N ratio of Lemna (Table 2) suggests that it undergoes rapid decomposition in the soil and can therefore be used as an organic fertilizer, applied either fresh or dried, without prior composting [46]. Compared to traditional green manures, the use of Lemna has the advantage of not leaving the soil unused, or leaving it unused for a shorter period of time when Lemna is pre-incubated in the soil before planting the crop. In our research, we adopted a 28-day pre-incubation period, as suggested by Chikuvire et al. [19]. In their study on Swiss chard, these authors demonstrated that, despite the low C/N ratio, this period yielded the best synchronization between nutrient release from Lemna and the crop’s nutrient demand.

3.2. Summer Growing Cycle

During the summer growing cycle, the climate was characterized by hot and dry conditions. As reported by the Copernicus Report [47], Europe experienced one of its hottest summer on records in 2022. In June, temperatures were above average across most of Europe, with southern countries being the most affected. In Tuscany, an average temperature of 3 °C and 2.6 °C above the mean of the thirty years 1991–2020 was recorded in June and July 2022, respectively.

Despite the choice of a variety (‘Pereion’) suitable for summer cultivation, and maintaining the field capacity of the soil, extreme environmental temperatures under the high tunnel used for the trial significantly reduced lettuce growth. The Urea-treated plants were the most affected, appearing stressed and showing signs of burning on the leaf margins within seven days after transplant. Since many of these plants died before harvesting and the surviving ones reached an insignificant head diameter (8 cm on average) and weight (16.6 g fresh weight on average), we decided to exclude the Urea treatment and compare the Lemna treatments only with the unfertilized Control. It is conceivable that what was observed with the Urea treatment can be attributed to an intense urease enzyme activity, which increases with increasing soil temperature up to 45 °C [48]. The high temperature recorded from the first week of cultivation could have led to a rapid hydrolysis of urea and the release of ammonium ions (NH₄⁺). As reported by Taylor et al. [49], high temperatures affect the kinetics of ammonium oxidation, and the nitrification process may not be fast enough to transform NH₄⁺ into nitrite (NO₂⁻) and nitrate (NO₃⁻), resulting in the accumulation of NH₄⁺ ions and the release of ammonia (NH₃) in the gaseous state. This process, also favored by the high humidity and alkaline pH of the soil, probably led to a toxic effect on the Urea-treated plants at both root and leaf levels.

The diameter of lettuce heads during the summer cycle, measured weekly, is reported in Figure 3a. Lemna-treated plants at the dose of 60 and 120 kg ha⁻¹ N did not differ from Control, while L180 showed smaller head sizes, with values significantly different from Control in the first three weeks. At harvest, the head diameter of lettuce was on average 14.8 cm, 16.6 cm, 17.0 cm, and 17.9 cm in L180, Control, L120, and L60-treated plants, respectively. As shown in Figure 4, no differences in fresh and dry weight, dry weight percentage, and number of leaves were observed between treatments at harvest. This result can be attribute to the large variability found among plants within the same treatment. As an average of all the treatments, the lettuce heads had a fresh weight of approximately 48.0 g per plant, a dry weight slightly above of 5.0 g per plant, and 26 leaves.

The effects of fertilization with duckweed species on the fresh and dry weight of different crops have been studied by other authors with conflicting results. In a greenhouse experiment, Fernandez-Pulido et al. [25] observed an increase in the fresh and dry weight of kale and sorghum treated with duckweed, but not, as we also found in lettuce, for beet and tomato. Mahofa et al. [18] obtained an increase in tomato yield using a mixture of duckweed, soil, chicken droppings, and sawdust. Chikuvire et al. [19] and Jilimane [20] observed an increase in spinach and Swiss chard dry weight using L. minor and Wolffia arrhiza as fertilizers, respectively. In accordance with our results for the summer-grown lettuce, Pratiwi et al. [26] did not find any effect of the administration of Lemna on the number of leaves of red spinach.

The dry weight percentage of lettuce ranged between 11% and 12% for all treatments (Figure 4). These values, much higher than those normally reported for this species (about 7%) [50], can be attributed to the stress conditions, such as high temperatures and low relative humidity of the air, to which the plants were subjected during cultivation. It is well known that stress conditions alter water absorption capacities, and promote cell wall thickening and secondary metabolites production (e.g., lignin) as a defense mechanism [51,52].

Leafy vegetables tend to accumulate high levels of nitrate in the leaves. Nitrate itself is not toxic to human health, but, after ingestion, it is endogenously transformed into nitrite and N-nitroso compounds, which can lead to severe pathologies [53,54]. For this reason, the European Union has set a maximum level of nitrate content allowed for the marketing of some leafy vegetables, including lettuce [55]. In our experiment, no differences in the nitrate content of lettuce were detected between treatments, and, on average, nitrate concentration levels were below the limit set by EU for lettuce grown in greenhouses in the spring–summer period (4000 mg kg⁻¹ f.w.) (Figure 5). However, it must be noted that for Control, L120, and L180, one of the three analyzed samples exceeded this limit (4087.1 mg kg⁻¹ f.w., 4113.5 mg kg⁻¹ f.w., and 4945.1 mg kg⁻¹ f.w., respectively).

Treatments did not influence the total N, P, or K concentrations in lettuce (Figure 5). This result is in contrast to those obtained in other species. Fernandez-Pulido et al. [23] reported that leaves of kale supplied with duckweed contained more N and P than those of the control plants. A positive effect of Lemna in terms of increasing plant tissue nutrient content was observed also by Kreider [56], Fernandez-Pulido [57], and Fernandez-Pulido et al. [23] in sorghum, and by Ahmad et al. [30] in rice.

The tested fertilization treatments were also evaluated in terms of NUE by means of an NUE indices calculation (

Table 4. Recovery in lettuce heads of the N supplied by the different fertilization treatments (cfr. Table 3) in the summer growing cycle. Data are means ± standard deviation (n = 3). Different letters in the same column show significant differences between means (Tukey Test); n.s.: not significant, * = different at p ≤ 0.05.

Treatment	PFP f.w. (g g−1)	NUEa f.w. (g g−1)	NUEp f.w. (g g−1)	PFP d.w. (g g−1)	NUEa d.w. (g g−1)	NUEp d.w. (g g−1)	REC (g g−1)
L 60	279.94 a	67.23	786.95	30.42 a	6.01	79.29	0.14
L 120	138.32 ab	31.96	654.22	14.99 ab	2.78	41.49	0.07
L 180	60.62 b	−10.29	−1976.62	6.94 b	−1.19	−139.75	−0.03
	*	n.s.	n.s.	*	n.s.	n.s.	n.s.

). The NUE indices are a well-established and widely used metric to assess plant responses to N availability [58,59]. Nitrogen is the most limiting factor for crop growth and production. Farmers usually apply large amounts of N to maximize yield and quality and to hedge against possible crop losses, often exceeding the actual crop demand, with serious consequences for the environment [60]. This behavior is especially common among vegetable growers, as vegetables are high-value crops wherein the cost of fertilization represents a smaller percentage of the total production cost [61]. The over-applied fertilizer rates, combined with both a shallow root system and a short cultivation cycle, explain the low use efficiency of N commonly observed in vegetables compared to arable crops [62,63]. In our experiment, the NUE analysis revealed a PFP index, calculated in both fresh and dry weight, that was higher in L60 than in L180 (Table 4), confirming that this index decreases with the increase in N supply [64]. The NUEa, NUEp, and REC were not significantly influenced by the N rate.

3.3. Autumn Growing Cycle

During the autumn growing cycle, the environmental conditions under the high tunnel used for cultivation were mild. The diameter of the lettuce heads progressively increased from transplanting to harvesting in each treatment, reaching at harvest 23.0 cm, 22.7 cm, 22.5 cm, 27.1 cm, 27.6 cm, and 27.7 cm for Control, Urea, COF, L60, L120, and L180, respectively. Differences between treatments were observed as early as the first week after transplanting, when L120 was higher than Control. Over the following weeks, no differences were observed between plants fertilized with different Lemna amounts, but Lemna-treated lettuce showed a significantly higher head diameter than Control, Urea, and COF-treated plants (Figure 3b).

Fernandez-Pulido et al. [25] compared the effects of fertilization with duckweed, inorganic fertilizer, and a mix of both, on the yield of beet, tomato, kale and sorghum. No significant differences in fresh and dry weight were observed between treatments for beet, tomato and sorghum, while in the case of kale a higher yield was obtained with inorganic fertilization. In our experiment, fertilization with Lemna resulted in a significant increase in the yield of lettuce compared to both the Control and the other fertilization treatments, regardless of the dose of Lemna used (Figure 6). As an average of the three doses, the fresh weight of heads fertilized with Lemna was 137.7 g vs. 91.7 g (+50%), 83.4 g (+65%), and 80.7 g (+71%) of Control, Urea, and COF, respectively. Compared with Control, fertilization with Lemna increased lettuce yield by 32% (L60), 39% (L120), and 42% (L180).

The Lemna-treated plants also showed significantly higher dry weights than plants fertilized with Urea or COF; no difference was observed between L60 and Control, or between Control, Urea, and COF. In terms of number of leaves, the best performing treatment was L180 (42 leaves per plant), which significantly differed from the Control and both mineral and organic N fertilization. Compared with COF, L60 and L120-treated plants also showed a significantly higher number of leaves (Figure 6). For all treatments, the head dry weight percentage values are consistent with those reported in the literature [50]. The L120- and L180-treated plants showed a slightly but significantly lower dry weight percentage than Control, Urea, and COF plants, indicating the greater tenderness of the leaf tissues (Figure 6).

No differences between treatments were detected in nitrate content (Figure 7). According to EU Regulation 1258/2011 [55], the maximum nitrate content allowed for lettuce grown in the greenhouse in the autumn–winter period is 5000 mg kg⁻¹ f.w. This limit was not exceeded in any of the lettuce samples analyzed. The minimum concentration was found in control plants (1291 mg kg⁻¹ f.w.) and the maximum in L180-treated lettuce (3002 mg kg⁻¹ f.w.). As also observed during the summer growing cycle, treatments did not influence the total N, P, or K contents of lettuce (Figure 7).

The results of chlorophyll and carotenoids analysis in lettuce heads (

Table 5. Chlorophylls and carotenoids contents in lettuce subjected to different fertilization treatments (cfr. Table 3) during the autumn growing cycle. Data are means ± standard deviation (n = 3). Different letters in the same column show significant differences between means (Tukey Test); n.s.: not significant, * = different at p ≤ 0.05.

Treatment	Chlorophyll a (μg mg−1 f.w.)	Chlorophyll b (μg mg−1 f.w.)	Chlorophyll a + b (μg mg−1 f.w.)	Carotenoids (μg mg−1 f.w.)
Controllo	0.847 ± 0.013	0.286 ± 0.021 ab	1.133 ± 0.034	0.244 ± 0.015
Urea	0.730 ± 0.022	0.246 ± 0.022 b	0.976 ± 0.024	0.209 ± 0.008
COF	0.769 ± 0.071	0.272 ± 0.025 ab	1.041 ± 0.095	0.223 ± 0.013
L 60	0.766 ± 0.032	0.255 ± 0.022 b	1.021 ± 0.053	0.215 ± 0.009
L 120	0.770 ± 0.052	0.270 ± 0.014 ab	1.040 ± 0.062	0.219 ± 0.021
L 180	0.835 ± 0.072	0.299 ± 0.022 a	1.133 ± 0.094	0.224 ± 0.021
	n.s.	*	n.s.	n.s.

) show significant differences in chlorophyll b content, with higher values in treatment L180 (0.299 μg mg⁻¹ s.f.) than in both L60 (0.255 μg mg⁻¹) and Urea (0.246 μg mg⁻¹). No significant differences were found for chlorophyll a, a + b or carotenoids. Pratiwi et al. [26] did not observe any effect on chlorophyll and carotenoids when treating red spinach plants with a Lemna-based liquid fertilizer, while an increase in leaf chlorophyll content in olive plants treated with aqueous Lemna extract was found by Regni et al. [65].

The statistical analysis of N recovery data has highlighted differences between treatments for the NUE components considered in this research except for NUEp (

Table 6. Recovery by lettuce heads of the N supplied by the different fertilization treatments (cfr. Table 3) in the autumn growing cycle. Data are means ± standard deviation (n = 3). Different letters in the same column show significant differences between means (Tukey Test); n.s.: not significant, *, **, and *** = different at p ≤ 0.05, 0.01, and 0.001, respectively.

Treatment	PFP f.w. (g g−1)	NUEa f.w. (g g−1)	NUEp f.w. (g g−1)	PFP d.w. (g g−1)	NUEa d.w. (g g−1)	NUEp d.w. (g g−1)	REC (g g−1)
Urea	413.79 ab	−40.89 bc	600.08	25.68 b	−3.03 b	29.46	−0.08 b
COF	400.46 ab	−54.22 c	−432.58	25.57 b	−3.14 b	−20.22	−0.08 b
L60	622.48 a	167.80 a	543.28	35.87 a	7.16 a	23.14	0.30 a
L120	345.88 b	118.53 ab	576.04	19.15 bc	4.79 ab	22.14	0.21 a
L180	245.13 b	93.57 abc	581.01	13.28 c	3.71 ab	22.97	0.16 ab
	**	*	n.s.	***	*	n.s.	*

). The L60 treatment showed no difference in PFP f.w. compared with the same dose of N given as Urea or COF. As Lemna was increased to an N dose of 120 kg ha⁻¹ and 180 kg ha⁻¹, PFP f.w. decreased significantly. The values of PFP d.w. revealed a greater N recovery from L60 than from all the other treatments, i.e., in both cases of the same dose of N provided as mineral fertilizer (Urea) or commercial organic fertilizer (COF), and in the case of higher Lemna amounts (L120 and L180). As stated by Dobermann [66], PFP is the most important component of NUE from the farmers’ point of view because it shows the effectiveness of converting the N supplied to the crop into yield. Therefore, in our experiment, L60 was the treatment with the best performance in terms of N productivity. Higher NUEa values (both on f.w. and d.w. basis) were observed with L60 than with Urea and COF, while no differences were found between Lemna treatments. The REC expresses the ability of the crop to absorb from the soil the N provided by the fertilizer [28]. The values of REC observed in this experiment were consistent with those reported for lettuce in previous studies [37,67,68,69], and confirm a low efficiency of this species in the recovery of N supplied with fertilization. However, a higher share of the N contained in the administered fertilizer was found in plants fertilized with L60 and L120 compared to Urea and COF. Thus, Lemna seems to have made the N available in a way more suited to the rate of uptake of the plants. Urea may have released the N too quickly, with consequent probable loss through leaching, while, on the contrary, COF may have released N too slowly compared to the needs of lettuce. This could also explain why fertilization with Urea and COF did not result in an increase in production compared to the unfertilized control (Figure 6).

4. Conclusions

Recycling plant waste as a nutrient source for crops represents a circular approach to enhancing the sustainability of intensive horticultural production systems.

This study was based on the hypothesis that L. minor, an invasive aquatic species that the farm would otherwise have to manage as waste, could be used as organic fertilizer. Specifically, we evaluated the suitability of Lemna for lettuce fertilization in two different growing seasons. In the summer growing cycle the extremely hot environmental conditions severely affected plant growth, and probably invalidated the effects of the fertilizer treatments. However, fertilization with Lemna did not show any negative effects on lettuce. In the autumn cultivation cycle, fertilization with Lemna showed better results than conventional inorganic and organic fertilizers, and increased yield compared to no fertilization. No differences in yield and quality were observed between the different levels of Lemna (corresponding to 60, 120, and 180 kg h⁻¹ N, respectively), but the lowest level showed a better NUE, suggesting this as the most suitable for the fertilization of lettuce. On the other hand, a greater soil amending effect and additional quantities of N possibly made available for subsequent crops—aspects not evaluated in this study—could be achieved by the application of higher Lemna levels. The dynamics of Lemna decomposition in the soil and its impact on nutrients availability and soil organic matter content are important issues to be addressed in further studies to have a more comprehensive understanding of the potential use of this weed as green manure.

Our findings suggest that Lemna biomass may be a suitable substitute for synthetic fertilizers with advantages for yield and no effect on quality. Thus, it was confirmed that the use of Lemna as an organic fertilizer can allow the farmer to profitably exploit waste products and, at the same time, reduce the cost of purchasing commercial fertilizer, with environmental and economic advantages.