1 Introduction

The importance of developing novel sustainable techniques able of enhancing crop yields while mitigating substantial losses has emerged as a crucial global issue (Dmytryk and Chojnacka 2018; Jägermeyer, 2020). To improve the quality and productivity of crops it is imperative to enhance nutrient uptake and utilization efficiency. Simultaneously, it is essential to fortify the inherent defence mechanisms of plants against pests and diseases, thereby reducing reliance on chemical interventions (Costa et al. 2019).

In this contest, any improvement in agricultural practices aimed to increase nutrient uptake could be of great interest to researchers and growers (Buono 2021). Among interesting new strategies, biostimulants play a key role, representing agents able to enhance plant yields, significantly reducing the cropping systems’ dependency on chemical fertilizers and pesticides (Bulgari et al. 2019; Claros Cuadrado et al. 2019). The function of these classes of products is mainly due to the diversity of sources of the raw materials and the complexity of the resulting products, which in most cases may contain many poorly characterized molecules (Rouphael and Colla 2020). Furthermore, biostimulants are considered not only environmentally friendly and cost-effective solutions to sustain agriculture, but also compete with synthetic products in terms of efficiency in enhancing plant growth (Mrid et al. 2021).

An interesting class of biostimulants for their relevance at economic and commercial level, as well as for their great versatility, is represented by microalgae-based products (Mata et al. 2010; Ronga et al. 2019; La Bella et al. 2022). Microalgae are ubiquitarians unicellular photosynthetic organisms able to grow both in marine and freshwater environments (Priyadarshani and Rath 2012) or even in wastewater, allowing in this way a reduction of the costs production (La Bella et al. 2022). These microorganisms can be easily used to produce a wide range of highly valuable metabolites such as proteins, lipids, carbohydrates, carotenoids, vitamins, and hormone-like substances utilizable in crop production (Priyadarshani and Rath 2012).

Microalgae biostimulants positively affect plant growth by enhancing water uptake, root and shoot growth, tolerance to abiotic and biotic stresses, protein content in plant tissues, and the activity of the enzymes involved in the main metabolic pathways, such as nitrogen assimilation, photosynthesis, and carbon cycle (Bulgari et al. 2015; Puglisi et al. 2020). Furthermore, microalgae biomass represents an interesting alternative to replace or integrate mineral fertilizers, leading to improve soil quality and increase crop productivity. Introducing these biomasses into the soil, the chemical properties of treated soil enhance and the biological activity of microflora boosts, thereby influencing the overall biochemical state of soil fertility (Sharma et al. 2021).

Recently, the relevance of several microalgae, among them Chlorella vulgaris and Scenedesmus quadricauda, as bioactive agents in the soil, and their ability to improve plant growth, made them interesting products for a sustainable approach to the cultivation process (Puglisi et al. 2022). Several studies have been carried out on the biostimulant effects of living microalgae. Barone et al. (2019) observed that living cells of C. vulgaris and S. quadricauda exert a biostimulant effect on tomato seedlings, growing in a co-cultivation microalgae-plant system in a hydroponic Hoagland solution. Similarly, Zhang et al. (2017) studied the simultaneous cultivation of Chlorella infusionum and tomato plants, by using a hydroponic system, with the inputs only for crop production, and showed interesting results both for crop and microalgae, producing low-cost microalgal biomass and providing benefits for plant growth. These effects may be associated with the large number of secondary metabolites produced by microalgae (Puglisi et al. 2020).

Moreover, the extracts from the microalgae C. vulgaris and S. quadricauda exert a biostimulant effect on lettuce growth, both through root drench and foliar application, increasing the growth parameters and improving the activity of several enzymes involved both in primary and secondary plant metabolism (Puglisi et al. 2022; La Bella et al. 2021).

Furthermore, another important prerogative to improve the sustainability of the crop production process is related to developing new substances able to reduce the fertilizers doses, often exceeding and causing several environmental problems, such as accumulation in soil and, subsequently, lixiviation of nutrient excess into groundwater (Sharma et al. 2022). The main nutrients that are leached are nitrates, and depending on the dosage of fertilizers, soil type, and plant cultivation, nitrate leaching, ranging from 70 to 250 kg ha− 1, may occur (Fragalà et al. 2023). These phenomena may have a serious impact on environment, human and animal health, and lead to eutrophication and environmental pollution.

Lettuce (Lactuca sativa L.) is a well-known food plant worldwide grown due to its use and is generally cultivated as an annual crop, requiring relatively low temperatures to prevent it from early flowering. It can suffer from numerous nutrient deficiencies, as well as being plagued by several pests, fungal, and bacterial diseases (Kim et al. 2016). Due to the importance of lettuce as a food crop, different studies have been carried out on this specie, testing several approaches to reach new sustainable green solutions to improve its production process (Mógor et al. 2018; Silambarasan et al. 2021; Santoro et al. 2021, 2023).

In the present work, our hypothesis was to evaluate the effectiveness of microalgae biomasses, retrieved from a phy-coremediation process, as new soil biostimulants to improve the productive performance of lettuce seedlings under greenhouse conditions with potted plants. Thus, modernising the traditional vegetative lettuce cultivation process, along with mitigating the environmental effects of nitrate leaching through the soil. In this regard, the present study aimed to explore the potential reuse of microalgae biomass (C. vulgaris, S. quadricauda, and Klebsormidium sp. K39), which were previously grown on urban wastewater, in order to make them useful for irrigation purposes. Microalgae cells were tested for their effects on plant growth, mainly focusing on the nitrogen metabolism of lettuce seedlings. Furthermore, the study aimed to assess the impact of the addition of C. vulgaris, S. quadricauda, and Klebsormidium sp. K39 on the biochemical fertility of the soil, by exploring the principal enzymatic activities of the soil related to the microorganism metabolism. Finally, the effect of microalgae biomasses on the rate of nitrate lixiviation through the soil was also evaluated.

2 Materials and Methods

2.1 Chemicals

Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (Missouri, USA) and Thermo Fischer Scientific Inc. (Oxoid, Limited, Hampshire, UK) and were of analytical grade or higher.

2.2 Microalgae Culture

The microalgae used in this study were Chlorella vulgaris ACUF863, Scenedesmus quadricauda ACUF581, and Klebsormidium sp. K39. C. vulgaris and S. quadricauda were originally provided by the Algal Collection of University Federico II of Naples (Italy), while Klebsormidium sp. K39 was obtained in the algal collection of the Department of Agriculture, Food and Environment (Di3A) (University of Catania, Italy) (La Bella et al. 2023).

All the species were previously cultivated for 46 days, until the reaching of logarithmic growth phase, in a growth chamber in a standard Bold Basal Medium, including the following components: KH2PO4 (17.5 g L− 1), CaCl2·2H2O (25 g L− 1), MgSO4·7H2O (75 g L− 1), NaNO3 (250 g L− 1), K2HPO4 (75 g L− 1), NaCl (25 g L− 1), Na2EDTA·2H2O (10 g L− 1), KOH (6.2 g L− 1), FeSO4·7H2O (4.98 g L− 1), H2SO4 (1 mL L− 1), and the trace metal solution contains H3BO3 (2.86 g L− 1), MnCl2·4H2O (1.81 g L− 1), ZnSO4·7H2O (0.222 g L− 1), Na2MoO4·2H2O (0.39 g L− 1), CuSO4·5H2O (0.079 g L− 1), Co(NO3)2·6H2O (0.0494 g L− 1) (Wang et al. 2014). The cultures were bubbled with air and were maintained with a photoperiod of 16 h on/off, and a light intensity of 100 µmol photons m− 2·s− 1 with a light source (PHILIPS SON-T AGRO 400) (Baglieri et al. 2016). The microalgal biomasses were centrifuged and the pellets were rinsed with distilled water until the conductivity level reached < 200 µS cm− 1 (Puglisi et al. 2018).

After the cultivation in purity, the microalgae were employed for urban wastewater treatment as described in detail by La Bella et al. (2023). Briefly, the pure harvested microalgal biomasses were used as inoculum (100 mg L− 1 of fresh microalgal biomass) to evaluate their remediation performances and their ability to grow on this substrate. After 60 days of cultivation in wastewater, the biomasses were collected and separated from the culture medium by centrifugation at 4000 rpm for 10 min, as described in La Bella et al. (2023).

2.3 Experimental Site and Plant Material

The experiment was carried in a greenhouse located in Sicily. The climate is semi-arid Mediterranean, with dry, warm summers and mild winters. The lettuce seedlings (Lactuca sativa L., cv Romana) were provided by a local nursery in Catania and were transplanted at the stage of four true leaves. The cultivation was conducted in plastic pots (15 × 15 × 10 cm), filled with 1 kg of local soil, which was previously analysed (Table 1). Soil texture was assessed using the pipette method, which involved determining the particle size classes categorized as clay, silt, and sand (Violante 2000). The soil was air dried, sieved at 2 mm, and analysed for various parameters including water holding capacity (WHC), moisture content, pH, electrical conductivity (EC), organic carbon, phosphorus, total nitrogen, potassium, and Cation Exchange Capacity (CEC). The procedures described by Puglisi et al. (2018) were followed to conduct these analyses. The results of the soil characterization are reported in Table 1.

Table 1 Physical-chemical properties of the soil used in the experimental trials
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Before transplantation, microalgal cells were mixed directly as fresh microalgal biomass in the soil in a single dose to obtain two different concentrations, 50 and 500 mg kg− 1 of soil (w w− 1), respectively, and each of them was used alone or mixed with standard mineral fertilization (MF). MF consisted of a commercial solid ternary fertilizer NPK, made of NH4NO3, KH2PO4, and KNO3. Mineral fertilization corresponded to the amount commonly used in regular practice for lettuce cultivation (Muscolo et al. 2022). This MF amount corresponded to the following quantities per pot: 58.30 mg kg− 1 of soil of NH4NO3, 81.66 mg kg− 1 of soil of KH2PO4, and 69.3 mg kg− 1 of soil of KNO3.

Treatments were summarized in Table 2.

Table 2 Experimental design of different treatments
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The seedlings were grown under greenhouse conditions for 45 days, maintaining temperature values between 16±2 and 25±2 °C as mean minimum and maximum values. Solar radiation varied between 700 and 1400 µmol m− 2s− 1, representing optimal conditions throughout the growing season. The crop was daily irrigated to avoid stressful water conditions and to maintain 50% WHC, by dripline sprinkler for the 40th days. Furthermore, to simulate rain events and evaluate the phenomenon of leaching of nitrates in groundwater, two supplemental irrigation treatments were carried out, applying an amount of water that was 1/3 greater than the water holding capacity (WHC) of the soil, 8 and 28 days after transplantation, respectively. The water lixiviated from the pots was collected and stored at − 80 °C until further analyses.

The experimental design was completely randomized and for each treatment, 5 replicates were performed in 5 independent pots.

At the end of the experimental period, all the plants were sampled, frozen in liquid nitrogen, and stored at − 80 °C for further analytical determinations.

2.4 Chlorophyll Content

The chlorophyll content of lettuce leaves was measured at the end of the experimental period, before the final plant harvest, using a portable SPAD-502. Readings were performed on fully expanded leaves, four on intermediate leaves, and two on recently expanded leaves (León et al. 2007). The tool provides a numeric dimensionless value and is used for leaf chlorophyll estimation in lettuce (Pennisi et al. 2019).

2.5 Physiological Parameters of Lettuce Seedlings

Lettuce plants were divided into roots and leaves and separately measured, recording for each sample fresh weight (FW), length, and number of leaves.

Dry weight (DW) was determined for each plant, placing a set of subsamples of roots and leaves in a driving oven at 105 °C until to reach a constant weight, and allowed to cool for 2 h inside a closed bell jar. All parameters were recorded on each plant.

2.6 Protein and Enzymes Extraction from Roots and Leaves

Total protein and enzymes extractions from root and leaf tissues, separately collected from each treatment and replicates, were performed following Kaiser and Lewis (1984). Briefly, frozen lettuce samples were finely ground using liquid nitrogen, and an extraction buffer, containing 0.1 M phosphate buffer pH 7.5, 1 mM EDTA, 2 mM dithiothreitol, and 1.5 w v− 1 insoluble polyvinylpyrrolidone, was added in 1:12 w v− 1 ratio to samples of roots and leaves. The crude extract was filtered and centrifuged at 13,000 rpm for 30 min at 4 °C, and the supernatant was collected and precipitated with (NH4)2SO4 at 55% of saturation. The total protein content was determined by the Bradford (1976) method, using bovine serum albumin (BSA) as a standard curve (from 2 to 10 µg mL− 1 protein), and expressed as mg protein g− 1 DW. Analyses were performed for each sample.

2.7 Plant Enzymatic Activities

Nitrate reductase (NRA) was determined as described by Kaiser and Lewis (1984). In each reaction mix in a final volume of 2 mL, 100 µL of fresh leaf protein extract or 200 µL of fresh root protein extract (as above described) was added to a mixture made of: 0.1 M potassium phosphate buffer pH 7.5, 1 mg ml− 1 NADH, and 0.1 M KNO3. The samples were incubated at 28 °C for 15 min, and then the reaction was stopped by adding 1 mL of 1% (w v− 1) sulphanilamide in 1.5 M HCl, and 1 mL of 0.02% (w v− 1) n-1-napthyl-ethylenediamine dihydrochloride solution. Afterwards, each sample was centrifuged at 500 rpm for 5 min to remove interfering matter. NRA activity was measured spectrophotometrically (Jasco V-730 UV–vis spectrophotometer), by recording the absorbance at 540 nm and was expressed as units of nitrite mg− 1 protein, using a standard curve of sodium nitrite.

All other enzymatic activities were performed on aliquots of precipitated total protein extracts obtained as above described. The extracts were previously centrifuged at 13,000 for 30 min at 4 °C, the supernatant was discarded, and the pellet was dissolved in the smallest volume possible of the appropriate buffer.

The glutamate synthase (GOGAT) activity of lettuce extracts was determined via the procedure reported by Avila et al. (1987). Each assay mixture, with a final volume of 1.1 mL, contained 25 mM Hepes-NaOH (pH 7.5), 2 mM L-glutamine, 1 mM α-ketoglutaric acid, 0.1 mM NADH, 1 mM Na2EDTA, and 100 µL of enzyme extract. Samples were read spectrophotometrically at 340 nm, monitoring NADH oxidation for 4 min, and the activity was expressed as nmol NAD+ min− 1mg− 1 protein, using a molecular extinction coefficient of 6220 L mol− 1cm− 1.

The glutamine synthetase (GS) activity was quantified through the method proposed by Cánovas et al. (1991) and was evaluated as transferase activity. For the reaction, in a final volume of 750 µL, 100 µL of enzyme extract solution was added to the assay mixture containing 90 mM imidazole-HCl (pH 7.0), 60 mM hydroxylamine (neutralized), 20 mM KAsO4, 3 mM MnCl2, 0.4 mM ADP, and 120 mM glutamine. The reaction tubes were incubated for 15 min at 37 °C, and next 250 µL of a mixture 1:1:1 of 10% (w v− 1) FeCl3·6H2O in 0.2 M HCl, 24% (w v− 1) trichloroacetic acid, and 50% (w v− 1) HCl was added to stop the reaction. The γ-glutamyl hydroxamate produced was quantified spectrophotometrically at 540 nm, and GS activity was calculated from a calibration curve prepared by plotting the change in absorbance against different concentrations of γ-glutamyl hydroxamate and was expressed as µmol γ-glutamyl hydroxamate min− 1mg− 1 protein.

2.8 Soil Enzymatic Activities

Soil samples were analysed after the sampling at the end of the experimental period, after the harvesting of the lettuce seedlings.

Total hydrolytic activity in the soil (1 g) was performed by monitoring fluorescein diacetate activity (FDA), according to Green et al. (2006), by measuring the absorbance at 490 nm. The fluorescein concentration hydrolysed by the soil enzymes was calculated using a fluorescein standard calibration curve.

The determination of soil dehydrogenase activity (DHA) (EC 1.1) was carried out as described by von Mersi and Schinner (1991), using 1 g of soil. The quantification of iodonitrotetrazolium formazan (INTF) was carried out spectrophotometrically, by measuring the absorbance at 464 nm. To determine the concentration of INTF in the samples of soils, a calibration curve was prepared using known concentrations of INTF as standard.

Acid (EC 3.1.3.2) and alkaline (EC 3.1.3.1) phosphomonoesterase activities, indicated as ACP and ALP, respectively, were assayed according to Tabatabai and Bremner (1969) and Eivazi and Tabatabai (1977), using 1 g of soil sample. The content of p-nitrophenol (PNP) released in the soil samples was spectrophotometrically determined at 400 nm and was calculated using a standard calibration curve, prepared by plotting the change in absorbance against different concentrations of p-nitrophenol.

Urease activity (URE) (EC 3.5.1.5) was carried out as reported by Kandeler and Gerber (1988), using 5 g of soil. Urease activity was measured spectrophotometrically recording the absorbance at 690 nm and was expressed as NH4+ released in the reaction, using a standard curve of NH4Cl.

ACP, ALP, DHA, and URE activities, as well as organic carbon content (C), were used to calculate the potential biochemical index of soil fertility (Mw), according to Kalembasa and Symanowicz (2012), using the following formula:

Mw = (ACP + ALP + DHA + URE × 10− 1) × %C.

2.9 Determination of the N-NO3 in Leached Water

The concentration of nitrate in leached water was measured as described in Fragalà et al. (2023), by extraction with 1 M KCl for 1 h. The extracted solution was then determined spectrophotometrically, recording the absorbance at 540 nm. To quantify the nitrate concentration a standard curve of NO3− was used.

2.10 Statistical Procedures

The collected data were subjected to a one-way analysis of variance (ANOVA). Means were compared using Fischer’s protected least significant difference (LSD) test (p ≤ 0.05). The calculations were carried out on Excel version 2019 (Microsoft Corporation, Redmond, WA, USA) and Minitab (version 16.1.1, Minitab Inc., State College, PA, USA).

3 Results

3.1 Physiological Parameters of Lettuce Seedlings and Chlorophyll Content

The impact of microalgae cells on lettuce physiological parameters (Fig. 1), such as root length, shoot height, root and shoot fresh and dry weight, and number of leaves, is reported in Table 3.

Fig. 1
figure 1

Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF) at the end of the experimental trial

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Table 3 Morpho-biometric parameters of lettuce seedlings
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Statistical analysis of data revealed significant differences (p ≤ 0.05) at both the root and the shoot levels among the treatments and the fertilized control (Table 3). However, the unfertilized control consistently exhibited lower morphobiometric results compared to all other treatments. Microalgae treatments showed a positive trend in the treated plants’ shoot fresh weight, especially Cv50 and Cv500 + MF, which exhibited 91% and 76% increases over the Ctrl + MF, respectively. Similarly, all tested microalgae significantly improved root growth compared to the Ctrl + MF. In detail, S. quadricauda at the concentration of 50 mg kg− 1 provided the best performance in terms of root fresh and dry weight. Noteworthy, it’s interesting to highlight the tendency of microalgae to positively affect the monitored root parameters in every treatment with respect to the Ctrl + MF.

As regards the chlorophyll status of the plants, monitored in the field (Fig. 2), showed no significant differences among treatments.

Fig. 2
figure 2

Effects of microalgae treatments on the SPAD index of lettuce plants. The values are means of data from 5 pots and three replicates each. Absence of letters means not significant differences. Error bars indicate the standard error of the mean

Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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3.2 Protein Contents

The effects of microalgae cells on protein content in both the root and the shoot of treated seedlings are showed in Fig. 3. Consistent with the morphobiometric parameters, protein contents in the unfertilized control plants (Ctrl– MF), both in root and shoot, were significantly lower compared to all other treatments (Fig. 3).

Fig. 3
figure 3

Total protein content in roots (A) and leaves (B) of lettuce seedlings subjected to microalgae treatments. Different letters indicate significance according to Fisher’s protected LSD test (p = 0.05). Error bars indicate the standard error of the mean Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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At the root level, treatments with only microalgae did not show significant improvements in protein content compared to Ctrl + MF, except for Cv50 and Cv500 (Fig. 3A). In detail, Sq500 + MF showed the highest increase (+ 50%) compared to the mean content of Ctrl + MF plants. Although with lower performance, even C. vulgaris, applied alone or in combination with mineral fertilization, Sq50 + MF, Kleb50 + MF and Kleb50 + MF significantly increased average protein content compared to Ctrl + MF.

Similarly, at the shoot level, the most significant increases in protein content were observed in plants treated with all the microalgae cells (at both concentrations) and mineral fertilization. Moreover, a significant increase was also observed in seedlings treated with only microalgae, except in Cv500 and Kleb50.

3.3 Enzyme Activities in Lettuce Seedlings

The effects of microalgae cells on enzymes related to nitrogen assimilation in lettuce seedlings are reported in Figs. 4 and 5. All microalgae associated with mineral fertilization showed an increasing tendency in the activity of nitrate reductase, glutamine synthetase, and glutamate synthase, both in roots and shoots, when compared to the Ctrl + MF. Regarding the unfertilized control, all measured activities exhibited significantly lower values than those observed in other treatments. This finding can be attributed to the absence of inputs during the experimental trial.

Fig. 4
figure 4

Nitrate reductase (NRA) activity (A), glutamine synthetase (GS) activity (B), and glutamate synthase activity (C) in roots of lettuce seedlings subjected to microalgae treatments. Different letters indicate significance according to Fisher’s protected LSD test (p = 0.05). Error bars indicate the standard error of the mean Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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Fig. 5
figure 5

Nitrate reductase (NRA) activity (A), glutamine synthetase (GS) activity (B), and glutamate synthase activity (C) in shoots of lettuce seedlings subjected to microalgae treatments. Different letters indicate significance according to Fisher’s protected LSD test (p = 0.05). Error bars indicate the standard error of the mean Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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Figure 4A shows the NRA activity measured in lettuce roots. The treatments with C. vulgaris, at both concentrations (50 and 500 mg kg− 1), associated with mineral fertilization produced the highest increases (50 and 66%, respectively) in the activity compared to fertilized control plants. A similar increase was observed in samples treated with S. quadricauda (50 and 500 mg kg− 1) and mineral fertilization (52 and 47%, respectively). Overall, there was a consistent positive trend in NRA activity, and plants treated with microalgae in combination with fertilization showed the highest increases, except for Kleb500 + MF. On the contrary, no significant improvement was observed in plants treated solely with microalgae cells compared to Ctrl + MF.

Likewise, in Fig. 4B, the trend of GS activity in roots is shown, which is like that observed for NRA. The activity was consistently higher in plants treated with microalgae and fertilization compared to Ctrl + MF plants, except for plants treated with only microalgae, where the activity levels did not differ from the Ctrl + MF. The main increases were observed in plants treated with Cv50 + MF and Cv500 + MF, Sq50 + MF and Sq500 + MF, and Kleb50, exhibiting increases ranking from 18 to 33% over the Ctrl + MF.

GOGAT activity is shown in Fig. 4C. As observed with previous activities, treatments with C. vulgaris and S. quadricauda in combination with mineral fertilization increased all the values of GOGAT activity compared to Ctrl + MF plants. Moreover, Kleb500 + MF also provided a significant improvement of the activity level compared to Ctrl + MF. The main increases were recorded in plants treated with with Cv50 + MF and Cv500 + MF (24 and 20%, respectively) and Sq50 + MF and Sq500 + MF (21 and 23%, respectively).

Regarding the activity levels recorded at the shoot level (Fig. 5), the addition of microalgae cells into the soil tendentially increased all enzymatic activities. Just as observed for root data, the formulations with microalgae and fertilization provided the main statistically significant improvements. In detail, the highest increase in NRA activity was achieved in plants treated with S. quadricauda at a concentration of 500 mg kg− 1 + MF, showing an increase of about 28% with respect to Ctrl + MF. Furthermore, Cv50 + MF, Cv500 + MF, Kleb50 + MF, and Kleb500 + MF reached statistically comparable values to the best treatment.

As shown in Fig. 5B and C, even GS and GOGAT activities were positively influenced by microalgae, although with varying degrees of enhancement. Specifically, the highest increases in GS activity were observed in plants treated with Cv500 + MF, Kleb50 + MF, and Kleb500 + MF increasing by approximately 15%. Similarly, as regard GOGAT, all the plants treated with microalgae alone or in combination with mineral fertilization showed significant enhancements compared to Ctrl + MF. In detail, the main improvement of the activity, a 26% increase compared to Ctrl + MF, was reached in plants treated with Kleb50 + MF.

The effects of living microalgae cells on the monitored soil enzymatic activities (FDA, DHA, ACP, APL, and URE) are reported in Figs. 6 and 7.

Fig. 6
figure 6

Fluorescein diacetate (µg FDA per g of soil). The values are means of data from 5 pots and three replicates each. Different letters indicate significance according to Fisher’s protected LSD test (p = 0.05). Error bars indicate the standard error of the mean Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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Fig. 7
figure 7

Dehydrogenase activity (µg INTF per g of dry matter in 1 h) (A), acid phosphomonoesterase activity (µg PNP per g of dry matter in 1 h) (B), alkaline phosphomonoesterase activity (µg PNP per g of dry matter in 1 h) (C), urease activity (µg N per g of dry matter in 2 h) (D). The values are means of data from 5 pots and three replicates each. Different letters indicate significance according to Fisher’s protected LSD test (p = 0.05), absence of letters means not significant differences. Error bars indicate the standard error of the mean Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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Regarding the unfertilized control soil, all enzymatic activities showed significantly lower levels than those observed in other treatments. This result can be related to the absence of inputs during the experimental period.

Fluorescein diacetate hydrolysis was increased by microalgae treatments, and significant differences among treatments were observed (Fig. 6). Soil FDA activity was strongly influenced by treatments with S. quadricauda at 50 and 500 mg kg− 1, and Kleb50 which resulted in the highest increases in activity levels of about 150, 173, and 106%, respectively, compared to the Ctrl + MF. Nonetheless, all other treatments positively affected the hydrolytic activity. Furthermore, treatments involving only microalgae cells exhibited noteworthy results, showing higher recorded activity levels compared to Ctrl + MF soil. These results suggest the beneficial role of these microorganisms in enhancing the total soil microbial activity, thus contributing to the overall improvement soil quality, even in absence of fertilizer inputs.

Concerning ACP activity (Fig. 7B), all treatments with S. quadricauda and Klebsormidium K39 showed significant increases compared to the Ctrl + MF soil, highlighting that the presence of microalgae cells may lead to an improvement in activity. Among the different treatments, Sq50 + MF, Kleb50 + MF, and Kleb500 + MF reached the highest increase (from 82 to 89%) in ACP activity. However, the treatments Cv50, Cv500 and Cv50 + MF did not show a significant increase in ACP activity compared to the Ctrl + MF soil.

Regarding ALP activity (Fig. 7C), all microalgae treatments were found to be effective in increasing the activity compared to the fertilized control soil, except for Cv50, Cv500, showing similar behaviour in terms of their impact on ALP activity. As observed for ACP, Sq50 + MF showed the highest increase (about 69%) in ALP activity. Furthermore, Cv500 + MF, Kleb500, Kleb50 + MF, and Kleb500 + MF displayed significant improvements in activity compared to Ctrl + MF, reaching levels comparable to those of Sq50 + MF.

In contrast to the results for the ACP and ALP activities, the addition of microalgae resulted in a higher URE activity in the treated soils compared to the Ctrl + MF soil for only four treatments. Specifically, Cv500, Cv50 + MF, Sq50 + MF, and Kleb50 achieved statistically significant improvements in URE activity compared to the fertilized control soil. Among these treatments, Sq50 + MF demonstrated the most substantial increase of approximately 82.5% in URE activity.

Finally, to assess overall soil fertility, the potential biochemical index of soil fertility (Mw) was calculated to include activities of ACP, ALP, URE, and DHA, as well as organic carbon content. Interestingly, Mw values were quite different between treatments, but in each case, Mw values were always higher than the Ctrl + MF (Fig. 8). The most efficient treatment proved to be Sq50 + MF (81.5% increase).

Fig. 8
figure 8

Biochemical index of potential soil fertility (Mw) in soils treated with living cells of C. vulgaris, S. quadricauda, or Klebsormidium sp. K39. The values were calculated using the following formula: Mw = (ACP + ALP + DHA + URE x 10 − 1) x % C, considering average values of activities Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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3.4 N-NO3 Content in Leached Water

Figure 9 displays the N-NO3 content in the leached water during the experimental trials. As expected, a higher N-NO3- amount in the first leachate was detected compared to the second one. In detail, the Ctrl + MF sample exhibited the highest average value overall, approximately 1600 mg/L. However, only specific microalgal treatments showed a significant reduction in N-NO3 content when combined with mineral fertilization. These treatments include Sq500 + MF, Kleb50 + MF, and Kleb500. On the other hand, when the microalgae were applied alone, only Cv500 appeared to slightly reduce the N-NO3 content in the leached water, compared to the unfertilized control sample.

Fig. 9
figure 9

Nitrogen form N-NO3 in leached water (first and second leachate) after two supplemental irrigation treatments, simulating rain events, during experimental trials. Different letters indicate significance according to Fisher’s protected LSD test (p = 0.05) Lettuce plants subjected to mineral fertilization (MF) with the microalgae at two different concentrations (50 and 500 mg kg-1): Chlorella vulgaris (CV50 + MF and CV500 + MF), Scenedesmus quadricauda (SQ50 + MF and SQ500 + MF) and Klebsormidium sp. K39 (Kleb50 + MF and Kleb500 + MF)

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4 Discussion

The massive use of chemical fertilizers over time has led to negative effects on environmental health. Therefore, in this study, sustainable and environmentally friendly alternative compounds were tested to improve plant growth in lettuce seedlings, one of the most common leaf vegetables in the Mediterranean area, and soil ecosystem health. Moreover, several studies were recently carried out to evaluate the effects of microalgae on a wide range of crops, among these C. vulgaris and S. quadricauda were largely tested, mostly as cellular extracts (La Bella et al. 2022). At this regard, a biostimulant effect was detected on lettuce plants treated with a C. vulgaris extract, both at root and shoot levels, with a concentration of 1 mg Corg L− 1 (Puglisi et al. 2022). Similarly, Barone et al. (2018) observed that C. vulgaris and S. quadricauda extracts were able to act as biostimulants in the early stages of sugar beet cultivation, improving root and plant growth. Conversely, limited attention has been given to the potential effects of Klebsormidium sp. K39, which was successfully employed for urban wastewater treatment using a no cost substrate (La Bella et al. 2023).

The effectiveness of microalgae as biostimulants, either alone or combined with conventional mineral fertilization, was assessed in lettuce cultivation: the use of microalgae-based products in agriculture might reduce the use of mineral fertilizers and other chemical products, improving, in the meanwhile, plant growth and sustainability of the process (Parmar et al. 2023). However, the high production costs associated with conventional microalgae biomass limit its widespread adoption. To overcome this hurdle, the microalgae used in this study were grown in urban wastewater, in order to achieve their phycoremediation, as described in La Bella et al. (2023), proposing a cheap productive methodology in the system of microalgae biomass production (Enzing et al. 2014). As reported by La Bella et al. (2023), the high levels of organic and inorganic compounds in urban wastewater can positively affect microalgae growth. Furthermore, the low concentration of microbiological pollutants and absence of heavy metals (La Bella et al. 2023) exclude the possible contamination risk in microalgae biomasses, leading to a final by-product that can be safely employed in agriculture, in the perspective of a sustainable and circular economy.

Unlike organic or mineral fertilizers, biostimulants have no mineral-nutrition effects on plant growth since their nutrient concentration is too low (Baltazar et al. 2021). This is well-confirmed by the low doses at which they are able to affect plant metabolism (Miller 2020).

Our results demonstrated that C. vulgaris, S. quadricauda, and Klebsormidium sp. K39, at both concentrations (50 and 500 mg kg− 1), combined with mineral fertilization positively affected plant growth (Table 1; Fig. 2), such as the protein contents (Fig. 3) and the plant enzymatic activities monitored (Figs. 4 and 5). Otherwise, microalgae alone were effective in improving plant development but determined only a general slight increase compared to the fertilized control. These results suggest the possibility of reducing chemical fertilization in lettuce cultivation, mainly due to the ability of microalgal biostimulants to optimise the nutrient uptake efficiency of plants. Interestingly, remarkable improvements were recorded at root levels: this is probably due to the modality of application of microalgae cells, leading to a major activity of the enzymes detected, providing, consequently, a major development of roots and a major content of dry matter.

The increases in the root apparatus resulting from microalgae applications may also have contributed to an increase in nitrogen uptake, as previously demonstrated by Ertani et al. (2009), who showed in corn cultivation that root applications of protein hydrolysates can enhance nitrogen assimilation through an increase in key enzymes (such as NRA and GS). Similarly, the positive effects of various biostimulant applications on plant nitrogen content, following an improvement in enzymes of nitrogen cycle, in several vegetable crops such as lettuce, radish, and red pepper have also been observed (Liu and Lee 2012; Tsouvaltzis et al. 2014).

NRA is an enzyme found in the cytosol of plant cells and, it is considered a critical point in the nitrate assimilation pathway (Tejada-Jimenez et al. 2019; Tischner 2000). It plays a basic role in reducing NO3− to NO2− and acts as a key component in plant nitrogen metabolism (Nemie-Feyissa et al. 2013). NRA is commonly recognised as the rate-limiting step in this pathway and can impact plant growth and development. Our findings indicate a significant increase in NRA activity in roots treated with mineral fertilization and microalgae, suggesting enhanced nitrogen assimilation, except for Kleb500 + MF. However, the improvements observed in the leaves were comparatively lower than those achieved at the root level.

Regarding GS and GOGAT, these enzymes are also considered key players in the process of incorporating ammonium into carbon skeletons, and assimilating it into organic forms such as glutamine and glutamate (Gupta et al. 2012). At root level, our results show that the treatments involving microalgae in combination with mineral fertilization significantly increased GS activity, as well as GOGAT activity, except for Kleb50 + MF, which showed values comparable to Ctrl + MF. Concerning at foliar level, our results show that treatments Cv500 + MF, Kleb50 + MF, and Kleb500 + MF significantly increased GS activity; as regard GOGAT activity, treatments involving microalgae in combination with mineral fertilization significantly increased activity level. These increases were associated with better growth of the seedlings and higher protein content. The positive effects of microalgae cells on key enzymes of nitrogen cycle in roots are consistent with previous studies, as found by Barone et al. (2019) in a co-cultivation system of tomato plants and microalgae C. vulgaris and S. quadricauda. These authors evaluated simultaneously microalgae growth in the presence of plant roots in the medium for crops as well as the effects of microalgae on tomato plants. Furthermore, our findings are consistent with several other studies, highlighting the involvement of nitrogen metabolism in the promotion of the growth of various crops using common products with biostimulant properties (Bulgari et al. 2015). Overall, microalgae treatments stimulated nitrogen cycle enzymatic activities, particularly at higher concentrations, both in roots and leaves. Notably, lettuce seedlings treated solely with microalgae exhibited enzymatic levels comparable to or slightly higher than those in fertilized control plants, suggesting the potential for reducing mineral fertilization in lettuce cultivation.

Various soil enzymes are involved in soil fertility characteristics, biological cycling, soil nutrient conversion processes, and overall soil quality. Furthermore, these enzymes can be used as indicators and sensitive methods to assess the effects of environmental pollutants, agricultural practices, ecological differences, vegetation type, and different soil properties (Nannipieri et al. 2012; Utobo et al. 2015).

Each soil has a specific level of enzyme activities, and the types and number of enzymes may vary depending on factors such as the quality and amount of harvest residues in the soil, as well as the type and amount of organic and inorganic fertilizers applied (Akça et al. 2015; Koc et al. 2018; Barone et al. 2019). However, it is worth highlighting the importance of soil enzymatic activities and exploring new sustainable solutions to enhance microbial activities in the soil. Our results demonstrated that microalgal biomasses positively impacted soil enzymatic activities, particularly FDA, ACP, ALP, and URE (Figs. 6 and 7). Previous studies have shown similar effects in soil enzymatic activities, following treatments with blue-green algae and digestate from anaerobically digested microalgae (Rao and Burns 1990; Namli et al. 2023). De Caire et al. (2000) also reported increased activities of extracellular enzymes in soil treated with microalgae inoculants, aligning with our findings.

FDA allows estimation of soil microbial activities (Liao et al. 2020). Our results indicate that native soil microbial populations were positively affected by microalgae treatments, as a notable increase in FDA hydrolysis was observed in soil samples treated with microalgae, both alone and in combination with mineral fertilization, compared to Ctrl + MF soil (Fig. 6). Barone et al. (2019) obtained similar results and observed an improvement in FDA activity in soil samples treated with living cells of C. vulgaris and S. quadricauda or their cellular extracts. Furthermore, the inoculation of microalgal cells further increased FDA hydrolysis, suggesting an improvement in the indigenous microbiota by bioinoculants, probably related to increased substrate availability that stimulates the metabolic activity of microbes in the soil.

DHA is an intracellular enzyme, and its action represents total microbial activity (Saha et al. 2019). However, contrary to what was observed in other soil enzymatic activities, DHA was not significantly affected by the addition of microalgae cells to the soil (Fig. 7).

ACP and ALP catalyse the hydrolysis of ester-phosphate bonds, leading to the release of phosphate, which can be taken up by plants or microorganisms (Nannipieri et al. 2012). Overall, ACP and ALP exhibited similar behaviour after the application of microalgal cells. Substantial increases in both enzymatic activities were observed with C. vulgaris, S. quadricauda, and Klebsormidium sp. K39 in combination with mineral fertilization (except for Cv50 + MF), leading to an elevated availability of phosphorous (Fig. 7). However, treatments containing solely microalgae did not show any significant differences compared to the Ctrl + MF soil. In general, our results suggest that ALP activity is more stimulated in quantitative terms of PNP released compared to ACP activity, and it is consistent with the pH values of the soils, which remained quite constant throughout the experimental period (data not shown). These findings are consistent with studies showing ALP predominance in neutral or alkaline soils and ACP activity in acid soils (Rawat et al. 2021; Rao et al. 2014).

URE is a potential factor for evaluating soil nitrogen content; indeed, it is a crucial soil enzyme that plays an important role in the hydrolysis of urea to ammonia and carbamic acid, which is further converted to ammonia and carbon dioxide through a chemical hydrolysis process (Sharma et al. 2022). In our study, soil URE was enhanced only by four treatments (Fig. 7). These findings are in accordance with a previous study of Barone et al. (2019), where an improvement in URE activity in soil samples treated with living cells of C. vulgaris and S. quadricauda or their cellular extracts was reported. Furthermore, Kwiatkowski et al. (2020) conducted a three-year study to assess the impact of organic agriculture on soil quality and found that the continuous application of organic manure had a positive influence on soil urease and dehydrogenase activities.

Overall, the positive effects of microalgae treatments on FDA activity, coupled with the relatively high sensitivity of ACP and ALP to treatments, indicate that microalgae soil inoculation has great potential to improve the indigenous microbiota and the release of inorganic phosphorus (orthophosphate) from organic phosphomonoesters (Alef 1995). Furthermore, increases in URE activity, although observed only in some treatments, indicate the potentiality of microalgal cells as a biostimulant for nitrogen cycling (Siczek and Lipiec 2016). On the other hand, the less pronounced improvement in DHA might be associated to the low potential of these treatments for the production of adenosine triphosphate through the oxidation of organic matter in the soil (Siczek and Lipiec 2016).

Concerning the Mw index (Fig. 8), it can be considered a very useful index as it takes into account all the enzymatic activities analysed in the present study, in order to establish the most effective treatment in terms of soil fertility. Our findings showed Mw values quite different among the treatments (Fig. 8), in particular it appears that C. vulgaris, S. quadricauda, and Klebsormidium sp. K39 are able to positively affect soil functioning compared to the Ctrl + MF, alone as well as in combination with mineral fertilization. The increased values of the Mw index indicate that microalgae cells have a positive impact on the biological or biochemical activity of the rhizosphere, as previously reported by Pii et al. (2015), determining as a consequence an improved crop growth. Our results are also in agreement with Barone et al. (2019), who observed higher Mw values in soils treated with microalgae cells and cellular extracts of C. vulgaris and S. quadricauda. Furthermore, it is noteworthy microalgae cells may positively improve Mw index at a low application dose compared to other substances (Kalembasa and Symanowicz 2012). Taking all these results together, it is possible to hypothesize that the direct use of microalgae cells into soil treatment, alone or in combination with mineral fertilization, may have a bioactive effect by inducing enzyme activities using small amounts of biomass.

Regarding the leached water, the maximum leaching of nitrate was taken place from the treatment with only mineral fertilization, while the least was recorded from treatments with only microalgae cells (Fig. 9). Statistically, among the treatments, the nitrate contents in the leachates from Cv50, Cv500, Sq500, and Sq500 + MF were significantly lower than the Ctrl + MF treatment by approximately 50%, thus suggesting that the presence of microalgae reduce the loss of nitrate from the soil. Probably, the microalgae biomass in the soil, increasing the soil enzymatic activities and therefore the soil microorganism, led to an increased use of nitrogen by microbiota, and consequently a lower nitrate lixiviation.

Overall, our findings are in agreement with Sharma et al. (2021), who observed that the application of microalgal biomass (Chlorella minutissima) to soil significantly reduced nitrate leaching compared to chemically fertilized treatment soil. Our experiment may be also supported by the long-term study conducted by Nguyen et al. (2013) on corn and soybean crops to determine the effect of poultry manure and chemical fertilizer on nitrate leaching. These authors found that the leaching loss of nitrate was less in poultry manure compared to urea ammonium nitrate. This hypothesis is further supported by Fragalà et al. (2023), who tested bioproducts from pre-treated municipal biowaste as soil biostimulants. The authors found that these bioproducts may improve plant growth, affect various metabolic pathways, and reduce the environmental impact associated with nitrogen leaching, thus reducing the loss of nitrogen through lixiviation in groundwater. Similarly, microalgae may reduce nitrate lixiviation by increasing the uptake of nitrogen from the plant, as well as the microbial community living in the soil. Indeed, all microalgae treatments resulted in an improved plant uptake of nitrogen from the soil, determining enhanced seedlings growth. This was supported by the increase in total protein content in the edible portion of plants (Fig. 3), as well as the improvements of the main plant enzymes involved in the nitrogen pathway (Figs. 4 and 5) and of the soil microbial activities monitored (Figs. 6 and 7), hence leading to a great reduction in the amount of leached nitrate in the groundwater. This hypothesis is supported by the evidence that increased nutrient uptake, such as nitrogen from the soil, is one of the main processes studied among the mechanisms of biostimulant products (Abbate et al. 2009; Puglia et al. 2021).

However, this study presents only preliminary results on the effectiveness of microalgae, and further trials are needed to confirm their practical effectiveness in reducing nitrogen loss in groundwater. Reduction in chemical fertilizers by the use of sustainable and eco-friendly compounds can bring about benefits for microbial communities and environmental health. In this regard, as the present study demonstrates, microalgae are able to improve yields and reduce, as a consequence, the doses of chemical fertilizers, maintaining a high standard of production. Furthermore, microalgae also seem to be able to reduce nitrogen loss through lixiviation, contributing to a global issue, as the reduction of environmental pollution is often related to common agriculture practices.

Future studies under varied conditions and crop types will provide further insights into the efficacy of microalgae, offering solutions to enhance yields and sustainability in crop cultivation processes. These efforts contribute to a gradual decrease in conventional fertilizer use, supporting global initiatives for environmentally friendly agriculture practices.

5 Conclusion

Increased awareness of resource scarcity, environmental protection, food safety, and nutrition has created a need for more sustainable and resource-intensive agricultural production systems. In this context, this study demonstrated that microalgal biomasses, produced using wastewater as a growth substrate, can be considered as sustainable alternatives to improve the lettuce cultivation process, seedlings development and soil quality.

The microalgal biomass obtained through the phycoremediation process has shown great potential as biostimulants in the agricultural field, either alone or in combination with mineral fertilization, thereby suggesting an advantageous reduction in the use of the latter in current practices. The application of microalgae as a soil biostimulant results in a higher or equivalent yield of lettuce plants compared to the standard application of recommended doses of mineral fertilizers. Furthermore, to the best of our knowledge, this is the first investigation of the effects of direct application of Klebsormidium sp. 39 cells into the soil. Overall, the results of the present study provide new insights into the use of microalgal products as sustainable solutions capable of improving crop productivity while reducing the environmental impact of conventional agricultural practices, thus confirming our initial hypothesis. However, further studies are needed to evaluate the potential long-term effects of microalgae biomass obtained after phycoremediation, on various crops.

In conclusion, this study underscores the potential of microalgal biomass as sustainable biostimulants for improving crop production and soil health. By elucidating the mechanistic links between microalgae application, plant growth, nitrogen metabolism, and soil microbial wellness, our findings contribute to ongoing efforts to develop environmentally friendly agricultural practices.