Improved Phosphorus Bioavailability in Lettuce Crop via Naganishia albida Inoculation of Wastewater-Derived Struvite

Abstract

Phosphorus (P) is a vital element for optimal crop growth and agricultural productivity. Struvite, a P precipitate obtained from wastewater, is recognized as a slow-release, low-solubility fertilizer. The objective of this study was to evaluate the impact of inoculation with the yeast Naganishia albida on P bioavailability using struvite and triple superphosphate (TSP) in lettuce (Lactuca sativa L.) plants. Struvite fertilization improved N and P assimilation by 14–28% and 12–27%, respectively, compared to TSP and increased soil soluble P by 50% more than TSP and 186% more than the control. Inoculation reduced oxidative stress by 40–44%, improved plant growth by 28% with struvite and 7% with TSP, and increased acid phosphatase activity by 52.7% and 78.1%, respectively, improving nutrient bioavailability. Struvite showed high P solubility in the soil, with only a 3% difference between inoculated and non-inoculated treatments. In addition, the combination of fertilizer and yeast had a synergistic effect, increasing enzyme activity up to 1.8 times for struvite and 2.3 times for TSP. The results highlight the potential of struvite as a recycled fertilizer and the effectiveness of integrating fertilization with microorganisms to improve agricultural efficiency, reduce environmental impact and promote sustainable management in the framework of the circular economy.

1. Introduction

Phosphorus (P) is a vital macronutrient for plant nutrition and growth, influencing metabolic processes, cell development, energy transport (ATP, ADP), photosynthesis, and respiration, etc. This nutrient is extracted from non-renewable mineral resources, making it one of the most limiting elements for crop production globally [1]. Additionally, the efficiency of P fertilizers is significantly constrained by soil processes, such as sorption and precipitation, with only 0.1% of the total P being available for plant absorption [2]. As a result, the excessive use of phosphate fertilizers has grown significantly over the years to ensure an adequate supply of P to plants, raising concerns about the long-term sustainability of this resource [3].

A sustainable solution for P management in agriculture has been the recovery and recycling of this nutrient from wastewater as a substitute for conventional fertilizers. In this context, struvite (MgNH₄PO₄·6H₂O) is a precipitate of phosphate, ammonium and magnesium that has proven to be an effective fertilizer, with a P content (about 12% dry weight) comparable to that of phosphate rock or other common chemical fertilizers (ranging from 7 to 18% dry weight) [4], and high purity (94–98%) [5]. One of its key characteristics is its low solubility in water (1–5%), making it a possible slow-release fertilizer capable of reducing nutrient loss through leaching [6], which is desirable for a more efficient use of P by plants.

Nevertheless, low struvite solubility, particularly in soils with high pH (~8.5), could lead to an excessively slow P release, which may negatively impact plant growth by limiting the uptake of P and other nutrients, reducing photosynthetic efficiency, and increasing the production of reactive oxygen species (ROS) [1]. Although struvite has been shown to be as effective as conventional fertilizers over the long term [6,7], the mechanisms of its dissolution and nutrient release may be affected by factors such as soil type (acidic or alkaline), plant species, particle size, and application rate [8,9], etc. Therefore, P availability in the soil directly influences crop productivity, and the use of efficient alternatives for P solubilization is a good approach to improve the use of non-conventional P sources, such as struvite.

To provide more efficient and sustainable P utilization by cropped plants, inoculation with microorganisms has gained attention as a viable and eco-friendly practice [1]. Particularly, within the large group of plant growth-promoting microorganisms (PGPMs), phosphate-solubilizing microorganisms (PSMs) can be highlighted, which can be used as bioinoculants due to their ability to stimulate plant growth by means of the solubilization of organic or inorganic P sources [10]. Specifically, some bacteria, fungi, cyanobacteria, mycorrhizal fungi, and actinobacteria contribute to the mineralization of organic P and the solubilization of inorganic P, which is otherwise unavailable to plants [1]. Yeast plays a key role in P solubilization through various biological mechanisms, such as the production of organic acids [11], the release of phosphatase enzymes [12] and the production of extracellular polysaccharides [13]. These processes help to increase the availability of this essential nutrient in the soil, facilitating its uptake by plants [12]. Within this group is the yeast Naganishia albida, and recent studies have explored its ability to enhance plant growth by inoculating crops, particularly in extreme environments [11,14].

In general, several studies have examined the ability of certain PSMs in soils [1,15], while others have compared the effects of different types of P fertilizers on plant growth [4,6]. Additionally, a recent literature review highlights the significant contribution of PSMs to P nutrition when used in combination with conventional fertilizers, particularly phosphate rock [10]. However, few studies have investigated the potential of PSM to increase P availability from new mineral sources, such as struvite, and their effect on plant growth [16]. Similarly, the phosphate solubilizing capacity of yeasts is poorly documented, despite their potential application as bioinoculants [17,18,19]. Furthermore, current studies on N. albida have mainly focused on its ecology and role in the environmental microbiota, while its phosphate solubilizing capacity and potential as a biofertilizer have received little scientific attention.

Therefore, the aim of this study is to evaluate the effect of N. albida inoculation on the solubilization and bioavailability of struvite P and its effect on the growth and development of Lactuca sativa L. (lettuce). N. albida is expected to act as an effective bioinoculant by improving the availability of struvite P, a slow-release P source, through solubilization mechanisms. This research can help improve efficiency in the use of chemical fertilizers and P in the soil, as well as contributing to more sustainable agricultural practices.

2. Materials and Methods

2.1. Struvite Characterization

The P precipitates were obtained from the Mapocho–Trebal wastewater treatment plant facility in Santiago, Chile (33°32′22″ S 70°50′15″ O). This plant employs conventional activated sludge technology together with thermal sludge hydrolysis and conventional mesophilic anaerobic digestion, and operates with two main process lines: water treatment and sludge treatment. In the water treatment line, the uncontrolled formation of precipitates in the pipes caused clogging that affected the efficiency of the process. As a result, these precipitates were considered as waste and were available for analysis in this study. The struvite samples were washed with distilled water to remove impurities and soluble salts. The solid samples were dried at 105 °C for 24 h and then sieved through a 150 µm sieve.

2.2. Inoculum Preparation

Three microorganisms with phosphate-solubilizing capability were analyzed, provided by the Metabolomics Laboratory, Department of Chemical Sciences and Natural Resources, University of La Frontera, Temuco, Chile. The PSMs analyzed were the yeasts Meyerozyma guilliermondii and Naganishia albida, and the rhizobacterium Burkholderia caledonica, isolated from the hyper-arid Atacama Desert in northern Chile [14,20]. It should be noted that these microorganisms were isolated from extreme environments and were available for research. In preliminary studies, they were selected as the only examples from the set evaluated that demonstrated the ability to solubilize phosphate from struvite (results are presented below). The microorganisms were cultured by inoculating 100 µL of each dilution into a specific medium. Luria–Bertani (LB) medium was used for bacterium, and Yeast Extract Peptone Dextrose (YPD) medium was used for yeasts. The cultures were incubated at 30 °C along with shaking at 120 rpm during seven days. After incubation, the cultures were dispersed using vortexing for 5 min and then centrifuged at 500 rpm, adjusting the optical density at 600 nm (OD₆₀₀) to 0.8 [21].

2.3. Phosphate Solubilization Capacity

The phosphate solubilizing capacity of the microorganisms was determined by inoculating 10 μL of the cultures onto NBRIP agar plates (National Botanical Institute’s Phosphate growth medium), with 0.5% (w/v) struvite as the sole P source. Struvite was dissolved in the medium on a hot plate at 35 ± 5 °C along with shaking for 1 h at pH 7. Additionally, Bromophenol Blue was added at 0.25% (w/v) [22]. The plates were incubated for 7 days at 30 °C in triplicate, with a control using sterile NaCl solution at 0.9% (w/v). The phosphate solubilization index (PSI) was determined by measuring the diameters of the halo (DH) and colony (DC) [23].

For the quantitative phosphate solubilization assay, the microorganisms were inoculated into 50 mL of liquid NBRIP medium with 0.5% (w/v) struvite [23]. Struvite was dissolved in the medium on a hot plate at 35 ± 5 °C along with shaking for 1 h at pH 7. The cultures were incubated at 30 °C for seven days at 120 rpm in the dark. A 3 mL aliquot was extracted, and the samples were centrifuged at 10,000 rpm for 10 min [23]. Samples were taken every two days to measure soluble P concentration using the molybdenum blue stannous chloride reducing agent method, according to Standard Method 4500-P. D [20,24]. The blue color developed was measured at 600 nm using a microplate spectrophotometer (EPOCH, BioTek Instruments, Inc., Winooski, VT, USA).

2.4. Experimental Design and Conditions for Lettuce Growth

A mixed factorial design with two factors was used in the experiment. The first factor was fertilization at three levels: no fertilization (control), chemical fertilization (TSP) and struvite fertilization. The second factor was inoculation at two levels: no inoculation (control) and inoculation with N. albida. This combination resulted in a total of six treatments. Each treatment had five replicates (n = 5 per treatment), giving a total of 30 experimental units (N = 30).

The growing substrate consisted of a mixture of peat moss and perlite (v/v 70:30%), and the mix was autoclave-sterilized at 121 °C for 60 min. In order to strictly control the availability of P from struvite, it was decided not to use any other type of soil that might contain additional P. Lettuce (Lactuca sativa L.) was used as a model crop. Seeds were sterilized in a 5% (w/v) NaClO solution for 5 min and washed twice with distilled water. Subsequently, seeds were sown in polystyrene trays for seedling production. After 20 days, the seedlings were transferred to 1 L pots containing 300 g of the substrate medium described above. At this time, the seedlings were inoculated for the first time near the roots with 1 mL of N. albida suspension (10⁶ cells). To ensure adequate colonization by the micro-organism, a second inoculation was carried out 15 days later, following the protocol described by Santander et al. [21].

The P fertilizer dose ranged from 25 to 100 kg P/ha [25]. The optimal dose was set at 50 kg P/ha, equivalent to 73 mg P/kg [7]. Fertilizers were dosed according to their total P content, ensuring that all pots received the same amount. The estimated percentage of phosphoric acid (P₂O₅) in water-soluble TSP was 48%, while the estimated P₂O₅ content in the precipitated struvite was 12%. To ensure precise weighing of the fertilizers based on P, both struvite and TSP were powdered (<150 µm). They were applied 2 cm below the surface of the top layer of the substrate and covered with 100 mL of water added to each pot [7,26].

The experiment was conducted over 47 days in a controlled greenhouse environment at the Department of Chemical Sciences and Natural Resources, University of La Frontera, Temuco, Chile. The greenhouse conditions were 21–25 °C temperature, 50–60% relative humidity, and a 14/10 h day/night photoperiod. To maintain soil moisture near field capacity, equal amounts of water were applied to each pot every two days [21]. Essential nutrients, except P, were provided through 50 mL of Hewitt’s nutrient solution (see Table S1 in Supplementary Materials) at the start of the experiment and during the second inoculation (15 days after transplanting) [27].

2.5. Plant Analysis

2.5.1. Biomass Production, Nitrogen, and Phosphorus Uptake

At harvest, plant samples were washed with deionized water, and fresh weight and dry weight (65 °C, 48 h) of the shoots and roots were determined [21,26]. Shoot and root tissue nitrogen (N) and P concentrations (mg/g) were obtained. Briefly, 0.5 g of dried, ground shoot and root material was digested in an acid mixture of H₂SO₄(96%)/C₇H₆O₃/H₂O₂(30%), (v/w/v: 100/6/1) [28]. The digestates were used for spectrophotometric determination of P using the molybdate-blue method at 880 nm [24]. Nitrogen concentration was determined through colorimetric analysis at 650 nm using the Epoch UV–visible spectrophotometer (BioTek, Winooski, VT, USA) [28,29].

2.5.2. Photosynthetic Traits

Photosynthetic variables were measured one day before harvest from the second youngest leaves of five plants per treatment using the Targas-1 system (PP Systems, Amesbury, MA, USA), following the user manual instructions. The analysis included transpiration rate (E: mmol H₂O/m²s), stomatal conductance (gs: mmol H₂O/m²s), photosynthesis rate (A: μmol CO₂/m²s), and internal CO₂ concentration in leaves (Ci: μmol CO₂/mol) [21].

2.5.3. Photosynthetic Pigments

Approximately 1 g of fresh tissue was stored and ground in liquid N₂ to obtain a fine powder, which was then stored at −80 °C for subsequent analysis of photosynthetic pigments [19]. Chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll content in leaves were determined using the method described by Lichtenthaler [30]. A 0.3 g sample of plant material was mixed with 3 mL of methanol–formic acid extractant (95/5%, v/v) in the dark. The samples were then sonicated at 130 W (Sonics and Materials, Newtown, CT, USA) for 60 s at 40% amplitude. Afterward, they were shaken for 30 min at 200 rpm and centrifuged at 4000× g for 10 min. The supernatant was measured at 652 nm and 665 nm using a Synergy H1 Hybrid Multi-Mode microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Chlorophyll concentrations were calculated using the following equations:

Chl a = 12.25*Abs 665 − 2.79*Abs 652

(1)

Chl b = 21.50*Abs 652 − 5.13*Abs 665

(2)

Chl total = 7.15*Abs 665 + 18.7*Abs 665

(3)

2.5.4. Identification and Quantification of Phenolic Compounds and Antioxidant Capacity

For total phenolic compounds and antioxidant activities, the supernatant from the pigment extracts (Section 2.5.3) was transferred to a separate tube, protected from light, and stored at −20 °C [14]. Total phenolic concentrations were determined using the Folin–Ciocalteu method adapted for a microplate reader [31]. Briefly, 15 μL of the extract, 750 μL of deionized water, 75 μL of Folin–Ciocalteu reagent, 300 μL of Na₂CO₃ (20% w/v), and 360 μL of deionized water were added. The mixture was incubated in the dark for 30 min at 20 °C. Absorbance readings were taken at 750 nm, with gallic acid solution used as the standard. Antioxidant activities were determined as Trolox equivalent antioxidant capacity (TEAC), copper-reducing antioxidant capacity (CUPRAC), and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity [21]. These activities were measured using the microplate spectrophotometer (EPOCH, BioTek Instruments, Inc., Winooski, VT, USA) at 734, 450, and 517 nm, respectively, using Trolox as standard.

2.6. Soil Analysis

2.6.1. Available Phosphorus

Soil samples from each treatment were collected and stored at −20 °C for further analysis. Soil pH was measured using a glass electrode in a suspension of soil-to-water at a ratio of 2:5. Available P concentrations were determined using the Olsen method [32]. For this, 0.5 M sodium bicarbonate (NaHCO₃) solution adjusted to pH 8.5 was used for extraction, with a soil-to-solution ratio of 1:20. Samples were shaken horizontally for 30 min at 180 rpm and then filtered through Whatman No. 45 paper. P content in the extract was quantified using the molybdenum blue method with ascorbic acid at 880 nm using Epoch UV–visible equipment from BioTek (Winooski, VT, USA) [24].

2.6.2. Enzymatic Activity

Rhizosphere substrate samples from the treatments were collected and stored at −20 °C for enzyme analysis. Phospho-mono-esterase activity was determined using the method proposed by Tabatabai and Bremner [33], which involves measuring the p-nitrophenol released by incubating 1 g of soil with 4 mL of 50 mM buffer and 1 mL of 5 mM p-nitrophenyl phosphate (PNP) substrate at 37 °C for 1 h. The buffer used for acid phospho-mono-esterase was sodium acetate (C₂H₃NaO₂) at pH 6.5, and for alkaline phospho-mono-esterase sodium bicarbonate buffer (NaHCO₃) at pH 10.5. After incubation, 1 mL of CaCl₂ (0.5 M) and 4 mL of NaOH (0.5 M) were added to stop the reaction. Samples were then centrifuged at 13,000× g for 15 min at 4 °C, and the supernatant was collected and colorimetrically measured at 410 nm using an Epoch UV-visible spectrophotometer (BioTek, Winooski, VT, USA).

2.7. Statistical Analysis

All results are presented as means of five replicates with standard deviation (SD). Statistical analyses were performed using R Studio software (Version 4.2.2-2022). Normality tests (Shapiro–Wilk test), variance homogeneity tests (Levene’s test), two-way ANOVA, and least significant difference (LSD) tests were conducted to compare means. Additionally, the data were subjected to principal component analysis (PCA) and Pearson correlation matrix analysis to evaluate the multivariate effects of inoculation and fertilization treatments as main factors. The statistical significance was established at p < 0.05.

3. Results

3.1. Phosphate Solubilization Capacity by Microorganisms

Table 1. Characteristics of phosphate solubilization from struvite as a phosphorus source by the different microorganisms studied.

Microorganisms	Phosphate Solubilization Index (PSI)	Phosphate Solubilization (mg/mL)	Final pH
Burkholderia caledonica	1.52 ± 0.28 ab	1079.17 ± 228.07 a	6.4 ± 0.07
Meyerozyma guilliermondii	1.58 ± 0.16 a	805.09 ± 131.99 b	6.3 ± 0.02
Naganishia albida	1.40 ± 0.07 ab	1186.57 ± 57.80 a	5.6 ± 0.10

Values are expressed as the mean ± SD. Different letters indicate significant differences (p ≤ 0.05) between treatment means according to LSD test.

shows the phosphate solubilization capacity of the tested microorganisms, which varied significantly. Among them, M. guilliermondii (yeast) and B. caledonica (rhizobacterium) exhibited the highest phosphate solubilization indices (PSI), with values of 1.58 ± 0.16 and 1.52 ± 0.28, respectively. Regarding the quantitative phosphate solubilization, the initial concentration of soluble phosphate in the culture medium containing struvite as the sole P source was 108.19 ± 10.32 mg/L, with an initial pH of 7.0. After 7 days of incubation, N. albida reached the highest solubilization level, with a concentration of 1186.57 ± 57.80 mg PO₄/L, significantly higher than that of M. guilliermondii, which solubilized 805.99 ± 131.99 mg PO₄/L. The presence of microorganisms in the struvite-containing culture medium led to a decrease in the pH of the supernatant. N. albida caused the most significant reduction, reaching a pH of 5.6 ± 0.10. In contrast, the other inoculated microorganisms did not cause notable acidification, reaching final pH values of 6.3–6.4.

3.2. Biomass Production

Table 2. Fresh and dry weight of shoots and roots of lettuce plants, non-inoculated or inoculated with Naganishia albida, and under different phosphorus fertilization treatments (Control, TSP and Struvite).

	Fresh Weight (g/Plant)		Dry Weight (g/Plant)
Treatment	Leaves	Roots	Leaves	Roots
Not inoculated
Control	35.35 ± 2.18	15.85 ± 1.40	4.76 ± 0.36	2.13 ± 0.21
TSP	36.42 ± 2.33	13.95 ± 0.55	4.58 ± 0.32	1.75 ± 0.10
Struvite	34.60 ± 3.13	12.13 ± 1.67	4.52 ± 0.38	1.59 ± 0.21
Inoculated
Control	46.98 ± 4.02	18.91 ± 1.20	5.08 ± 0.19	2.07 ± 0.24
TSP	39.25 ± 1.72	14.46 ± 2.38	5.04 ± 0.20	1.84 ± 0.22
Struvite	44.46 ± 1.31	14.28 ± 0.62	4.47 ± 0.46	1.44 ± 0.21
Significance
Fertilization	ns	***	*	***
Inoculation	***	**	*	ns
Fertilization × Inoculated	***	ns	ns	ns

Values are expressed as the mean ± standard deviation. Significance: ns = not significant; * significant at p < 0.05; ** significant at p < 0.01; *** significant at p < 0.001.

shows the biomass production of fresh weight (FW) and dry weight (DW) in leaves and roots. Biomass production, both fresh weight (FW) and dry weight (DW), in shoots and roots was significantly influenced by the experimental factors used. Fresh weight (FW) was notably affected by inoculation with N. albida, both in the shoots and in the roots (p < 0.001). On the other hand, fertilization had a significant impact on root biomass production, both in FW and in DW. However, only the FW of the shoots was influenced by the interaction of the two factors. The highest biomass was observed in the inoculated treatments, particularly in the non-fertilized treatment, with a total biomass of 65.89 ± 4.18 g/plant in FW and 7.15 ± 0.39 g/plant in DW. The lowest values were observed in the treatments fertilized with struvite, regardless of inoculation.

3.3. Nitrogen and Phosphorus Uptake

Table 3. Nitrogen and phosphorus uptake in shoots and roots of lettuce plants, non-inoculated or inoculated with Naganishia albida, and under different P fertilization treatments (Control, TSP and Struvite).

Treatment	Nitrogen (mg/Plant)		Phosphorus (mg/Plant)
	Leaves	Roots	Leaves	Roots
Not inoculated
Control	41.03 ± 4.90	9.35 ± 1.15	29.82 ± 2.88	11.96 ± 1.48
TSP	47.27 ± 7.80	8.01 ± 0.75	34.67 ± 3.72	12.73 ± 2.34
Struvite	54.38 ± 8.73	8.18 ± 0.63	41.27 ± 3.97	14.73 ± 3.21
Inoculated
Control	55.54 ± 3.76	9.64 ± 1.14	37.66 ± 2.86	10.50 ± 2.08
TSP	60.69 ± 2.49	8.74 ± 1.04	42.50 ± 3.86	14.75 ± 2.95
Struvite	74.62 ± 6.95	7.74 ± 1.18	44.41 ± 8.31	13.27 ± 2.88
Significance
Fertilization	***	*	***	ns
Inoculation	***	ns	***	ns
Fertilization × Inoculated	ns	ns	ns	ns

Values are expressed as the mean ± standard deviation. Significance: ns = not significant; * significant at p < 0.05; ** significant at p < 0.01; *** significant at p < 0.001.

presents the nutrient uptake in leaves and roots of lettuce plants. Nutrient uptake by shoots and roots of lettuce plants was significantly influenced by both fertilization and inoculation. However, no significant interaction was observed between the two factors, either in the leaves or the roots. Plants fertilized with struvite exhibited the highest values of N and P in the shoots, with significant improvements compared to both the control and TSP treatments, regardless of whether they were inoculated with N. albida. In the non-inoculated treatments, struvite increased N uptake by 14–28% and P uptake by 12–27%. In the inoculated treatments, the increases were 20–30% for N and 5–16% for P.

On the other hand, N and P content in the roots of lettuce plants showed no statistical differences when compared to fertilized and inoculated plants. However, a higher absorption of P than N was observed in the roots. For N uptake in the roots, the control treatment showed a value of 9.35 ± 1.15 mg/plant while, for P, both struvite and TSP fertilized plants reached 14.73 ± 3.21 mg/plant and 14.75 ± 2.95 mg/plant, respectively (Table 3).

3.4. Photosynthetic Traits and Pigments

Photosynthetic characteristics, including E, gs, A, and Ci, were measured (Figure 1). Both transpiration rate and stomatal conductance were strongly influenced by fertilization. Transpiration showed a positive correlation with gs, with higher values observed in the inoculated treatments compared to the non-inoculated (Figure S1, Supplementary Materials). Moreover, fertilization with TSP produced a transpiration rate of 1.29 ± 0.10 mmol H₂O/m²s in the inoculated treatment and a gs of 112.16 ± 12.08 mmol H₂O/m²s. The photosynthetic rate was significantly influenced by fertilization, inoculation, and the interaction between both factors. In this case, photosynthesis showed a negative correlation with Ci, which was affected by the type of fertilizer: −0.2 for the control, −0.3 for TSP, and −0.7 for struvite (Figure S2, Supplementary Materials). The highest photosynthetic rate was observed in the non-inoculated struvite treatment, with a value of 7.27 ± 0.62 μmol CO₂/m²s. No significant differences were found in internal CO₂ concentration between the inoculation effect and the type of fertilization.

Similar patterns were observed in the contents of Chl a, Chl b, and Chl total (Figure 2). Inoculated plants showed 15% higher contents for Chl a and total Chl, and 18% higher Chl b compared to non-inoculated plants. Fertilization did not show significant differences in Chl a content; however, significant effects were observed for Chl b and Chl total. Treatments with TSP and struvite yielded similar results, with an increase of 2.3–4.4% compared to the control. In contrast, differences of 15% in Chl b and 18% in Chl total were observed relative to the control. No significant differences were observed in the interaction between inoculation and fertilization on these results.

3.5. Quantification of Phenolic Compounds and Antioxidant Capacity

Antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (Figure 3a), the Trolox equivalent antioxidant capacity (TEAC) method (Figure 3b) and the copper-reducing antioxidant capacity (CUPRAC) method (Figure 3c), and total phenols determined by the Folin–Ciocalteu method (TPC) (Figure 3d). All antioxidant methods showed a decrease in plants inoculated with N. albida compared to non-inoculated plants. However, they were only significant for struvite treatment. The values were 40–44% lower for DPPH, TEAC, and CUPRAC, and 27% lower for TPC in comparison with non-inoculated plants. For the DPPH method, the inoculated struvite treatment showed significantly lower activity (11.12 µmol/g FW) compared to the other treatments. Similar results were observed for TEAC, CUPRAC, and TPC, where the inoculated struvite treatment exhibited lower values; however, no significant differences were found with regard to the type of fertilization, whether for inoculated or non-inoculated plants.

3.6. Soluble Phosphorus and Soil Enzymatic Activity

The analysis of available P content in substrates across various treatments reveals significant effects of inoculation, fertilization, and their interaction (Figure 4). Struvite notably increased the available P content in the soil, achieving values of 187.04 ± 27.50 mg/kg in the non-inoculated treatment, and 181.60 ± 19.03 mg/kg in the inoculated treatment. When compared to TSP, struvite enhanced soil P levels by 50.3% and by 186.3% relative to the control group. Furthermore, the inoculated TSP exhibited a soluble P content that was 2.38 times greater than that of the non-inoculated treatment, which was significant.

Figure 5 shows the enzymatic activity of acid and alkaline phospho-mono-esterases in the rhizosphere of lettuce plants subjected to different treatments. Acid phosphatase (AcP) showed higher enzymatic activity, reaching 121.79 ± 42.52 µmol PNP/gh, compared to alkaline phosphatase (AlP), which recorded 60.44 ± 26.41 µmol PNP/gh, considering all treatments. The activity of AcP was influenced solely by inoculation, whereas AlP demonstrated greater sensitivity to variations in fertilization, inoculation, and their interaction. Notably, AcP activity was highest in the treatment with TSP, particularly within the inoculated treatment, which reached 164.41 ± 32.37 µmol PNP/gh; however, no significant differences were observed based on the type of fertilization. For control, TSP and struvite, the inoculated treatments showed an increase of 38%, 52% and 78%, respectively, over the non-inoculated treatments. In contrast, AlP activity remained similar across treatments, with the exception of the inoculated TSP treatment, which exhibited a higher value of 98.07 ± 8.87 µmol PNP/gh.

3.7. Multivariate Analysis

A principal component analysis (PCA) was conducted separately for inoculation and fertilization factors (Figure 6). The PCA revealed that the first principal component (PC1) accounted for 35.5% of the total experimental variance, while the second principal component (PC2) accounted for 20.5%. In PC1, the variables related with N absorption (0.31), P absorption (0.30), Chl a content (0.27), Chl b content (0.34), total chlorophyll (0.30), and photosynthesis as E (0.28) and A (0.29), which showed a positive correlation associated with plants inoculated with N. albida (Figure 6a). In PC2, the variables with the highest contributions were the antioxidant activities expressed as TPC (0.42), DPPH (0.43), CUPRAC (0.38), and TEAC (0.42), which were associated with the group of non-inoculated plants. Regarding fertilization, treatments with TSP exhibited a stronger association with plant growth-related variables, such as nutrient absorption and chlorophyll concentrations in PC1 (Figure 6b). Conversely, treatments with struvite addition displayed greater dispersion and were primarily associated with variables related to antioxidant activities, enzymatic activity, and available P. Additionally, a negative correlation was observed relative to FW (−0.25), indicating that as antioxidant activities increase, biomass production tends to decrease.

A Pearson correlation matrix was performed for all studied variables, both in inoculated treatments (see Supplementary Figure S1) and fertilized treatments (see Supplementary Figure S2). Inoculation strengthened the relationships between antioxidant activities and growth variables, such as DW, with correlations ranging from 0.3 to 0.5. Similarly, fertilization demonstrated a significant relationship with FW, particularly highlighting the effect of struvite, which improved correlations but in an inversely proportional manner (0.7 to 0.8). In the TSP treatment, a positive correlation was observed between chlorophyll content and nutrient absorption, with values between 0.5 and 0.9 (see Supplementary Figure S2b). Overall, fertilization increased P availability in the soil, showing correlations of 0.8 for TSP and 0.4 for struvite, compared to the control treatment, which exhibited a negative correlation of −0.3.

4. Discussion

The results obtained for phosphate solubilization from struvite demonstrated that yeast N. albida exhibited the highest solubilization, surpassing 1000 mg PO₄/L. These values were higher than those reported by Jokkaew et al. [23], where the bacterium Bacillus megaterium dissolved phosphate from struvite up to 835.4 mg PO₄/L whereas, with hydroxyapatite, it only reached 32.5 mg PO₄/L. The presence of N. albida in the medium showed a decrease in pH from 7.0 to 5.6. Previous studies show that the change in pH of the medium is closely related to the phosphate solubilizing capacity of the yeasts [11,19]. This decrease in pH helps to dissolve the P present in struvite and convert it into soluble and plant available forms, so they are closely related. Raklami et al. [11] evaluated the phosphate solubilization capacity of Naganishia sp. using complex inorganic phosphate, where they observed an acidification of the medium (pH 4–5). This result suggests that the solubilization process could be attributed to the production of organic acids, such as citric acid, lactic acid, oxalic acid and malic acid.

Organic acids, being low-molecular-weight compounds, can chelate the cation bound to phosphate through their hydroxyl and carboxyl groups, lowering the rhizosphere pH via gas exchange (O₂/CO₂) and the proton-bicarbonate equilibrium, thus releasing the associated P [15]. For struvite, the organic acids may have chelated the magnesium, increasing the P availability, as demonstrated by Jokkaew et al. [23]. However, this is not the only P solubilization mechanism employed by PGPMs; P solubilization could also occur through the release of siderophores, excretion of extracellular enzymes, or substrate degradation via mineralization [13,15].

Several studies have shown that yeasts play an active role in facilitating P supply to plants, directly correlating with improved plant growth [20]. The results obtained in this study indicate that inoculation with N. albida enhanced lettuce leave growth by 28% in treatments with struvite and control, and only 7% with TSP in terms of fresh weight. In terms of dry weight, increases were 16–17% for struvite and the control, while for TSP, this was only 4%. Hernández Jiménez et al. [16] demonstrated that combining struvite with Bacillus megaterium increased total DW by an average of 39%.

Fertilization showed a significant effect on root FW and DW, with the control outpacing the TSP and struvite treatments by 20% and 11–35%, respectively. These results contrast with previous studies by Vetrano et al. [34] and Sun et al. [35], who reported a positive fertilization effect on plant biomass. Vetrano et al. [34] observed a 30% increase in FW with 1 g/L fertilization compared to the control and also noted that DW was mainly influenced by fertilization rate. However, other studies have shown no significant biomass differences between different types of fertilizers [5,7,36]. For instance, Rech et al. [36] found no significant difference in root DW between struvite and TSP treatments in wheat crops, with a 16–25% variation. In this study, several factors that may have influenced the different outcomes include the type of crop, soil characteristics, and fertilizer dosage, which could explain the variations observed in different studies.

Additionally, our results indicated that both inoculation and fertilization had a positive effect on nutrient assimilation by plants [1]. In detail, struvite increased its P leave concentration by 28% compared to the control, and this increase was even greater with inoculation, reaching a 58% increase. These findings are consistent with those reported by Consentino et al. [37], who showed that inoculation with PGPMs, combined with N fertilization, increased leave nitrogen concentration by 47%. In similar studies, Yang et al. [8] confirmed that N absorption from struvite is comparable to that from N-based fertilizers such as urea and urea + TSP. In this sense, a notable advantage of struvite as a fertilizer is that it not only provides P but also N [4].

On the other hand, N. albida showed a high capacity to solubilize P; in the inoculated treatments, there were no differences between struvite and TSP, positively affecting fertilizer use efficiency. It has been demonstrated that bacteria and yeasts enhance their ability to solubilize and mineralize P through the production of various organic and phenolic acids, significantly altering the structures responsible for nutrient and water absorption [1,15]. Manzoor et al. [38] showed that applying PGPMs with rock phosphate (RP) as fertilizer increased P assimilation by 49%, compared to the sole use of RP.

Similarly, Hernández Jiménez et al. [16] reported a 33% increase in P assimilation when combining commercial struvite with the P solubilizing bacterium Bacillus megaterium in Avena sativa cultivation. In the non-inoculated treatments, struvite showed a high fertilizer capability, with a 13% increase in P assimilation in leaves compared to TSP. Although struvite has been reported to be less soluble and to release P slowly [5], our results differ from other studies that have demonstrated higher efficiency in P assimilation compared to conventional fertilizers [6,39,40]. Sun et al. [39] observed a significant disparity in P absorption in maize, with struvite increasing assimilation by 61%, while diammonium phosphate (DAP) achieved 167% compared to the control. However, for other crops, such as Chinese cabbage and cowpea, no notable effects were observed.

When comparing struvite with alternative fertilizers such as organic manure, Ryu et al. [41] evaluated the performance of organic manure, compost and wastewater-precipitated struvite. Their results showed that the P concentration in dried Chinese cabbage was 103% higher with struvite than with organic manure and 97% higher than with compost. Similarly, another study investigated the effect of biochar and soil microbial interactions on cabbage growth and found that the combined application of 2% biochar and microbes increased available P by 45.5% compared to soil treatments with or without biochar alone [42].

In other related studies, Cabeza et al. [4] demonstrated that struvite is as effective as TSP in acidic and neutral soils, while hydroxyapatite was only effective in acidic soils. This behavior varies among the different commercial sources of struvite; products derived from wastewater, such as Crystal Green, Berliner Pflanze, and BIOSTRU, have shown superior solubility and efficiency compared to conventional fertilizers, such as RP, single superphosphate (SSP), DAP, and TSP [25]. These results highlight that, although some studies have found no significant differences between different fertilizers [36,39,40], factors such as the specific type of fertilizer used, soil conditions, and crop type can considerably influence the overall efficiency observed in P solubilization from struvite.

Photosynthetic activity plays a fundamental role in agriculture, particularly in improving crop productivity and biomass [43]. Fertilization and inoculation have a positive effect on photosynthetic activity, as other research has shown [12,44]. On the one hand, excessive accumulation of P can be toxic while, on the other hand, insufficient supply can lead to reduced photosynthesis and hinder various physiological processes [45]. Fertilization with TSP and struvite favored transpiration rate and stomatal conductance, which showed a direct correlation. When there is an adequate supply of P, plants can maintain more open stomata (higher conductance), which favors photosynthesis and transpiration without compromising their water use efficiency [45].

In addition, high stomatal conductance favors CO₂ exchange for photosynthesis [43], which is reflected in our study. P also improves photosynthetic efficiency by increasing the rate of CO₂ fixation [45]. However, inoculation showed a greater influence on photosynthetic rate, with a correlation of 0.9, compared to P supply in the non-inoculated treatment, which showed a correlation of 0.4 (Supplementary Materials, Figure S1). In the case of struvite, no significant differences were observed between the N. albida inoculated and non-inoculated treatments. This is because the P present in struvite is already bioavailable and therefore does not require additional solubilization, unlike TSP. The Ci of CO₂ was not directly affected by fertilization or inoculation, indicating that the CO₂ available in the leaves was not a limiting factor in any of the treatments.

Photosynthetic pigments did not directly affect photosynthesis, but inoculated treatments showed better results compared to the control. In general, microbial inoculation favors an increase in chlorophyll concentration by improving nutrient bioavailability, stimulating plant growth and helping to alleviate stress conditions [14,17]. Regarding fertilization, a negative correlation with soil soluble P was observed for the control (−0.8) and struvite (−0.6, −0.4) treatments, while this was positive for TSP (0.5–0.9) (Supplementary Materials, Figure S2). A soil with higher P availability promotes higher chlorophyll production and more efficient photosynthesis [45].

No significant differences were found in antioxidant activity due to the type of fertilizer (control, TSP, or struvite). This suggests that the specific type of fertilizer does not significantly affect plant antioxidant capabilities, as inoculation does. Antioxidant activities (DPPH, TPC, TEAC, CUPRAC) showed an inverse relationship with variables related to plant growth and chlorophyll content, indicating a negative correlation between antioxidant activity and plant growth. The antioxidant capacity of a plant serves as a crucial measure for the assessment of its defense system, reflecting the plant’s response to stress. An increase in antioxidant activity is indicative of the plant’s response to elevated stress levels, whereby its antioxidant defenses are activated to provide protection [14,44].

Some studies have reported that inoculation with microorganisms improves oxidative activities by influencing several metabolic and physiological processes related to the production of antioxidant metabolites [15,44]. For example, Parada et al. [31] evaluated the effect of inoculation with the fungus Claroideoglomus claroideum on strawberry plants under different types of fertilization (chemical and organic). They found that the antioxidant activity was higher in treatments with 100% chemical fertilization and inoculation. However, in treatments with both chemical and organic fertilization at 50%, the best results were observed in plants without fungal inoculation.

In contrast, Santander et al. [46] observed that, in lettuce plants inoculated with arbuscular mycorrhizal fungi, non-enzymatic antioxidant activities, such as phenolic compounds, were lower compared to in non-inoculated plants. These results coincide with those of our research, where all inoculated treatments presented lower levels of antioxidant activity, especially in the treatment with struvite, which presented significant differences. The results indicate that plants inoculated with struvite show lower oxidative stress, probably due to N supply. Furthermore, a negative correlation was identified between antioxidants and N supplied by struvite (−0.8, −0.7) (Supplementary Materials, Figure S2c), suggesting that metabolic resources may be allocated towards plant growth and development, particularly in the absence of stressors, such as drought, salinity, or pathogens.

Soil P availability directly influences crop productivity [1]. Struvite treatment significantly increased the bioavailable P content in the rhizosphere soil. Notably, no solubilization effect was observed in the use of N. albida, suggesting that the constituent salts in the precipitate are sufficiently soluble to respond to other mechanisms, such as root exudates. However, a common source, such as TSP, considered highly soluble, still shows dependence on P release through the use of the solubilizing microorganism, which remains an interesting alternative to improve P use from less soluble sources.

Our results are in contrast to other research, where chemical fertilizers have a higher bioavailability [5,35,36]. Sun et al. [39] investigated the effects of struvite, diammonium phosphate (DAP) and a mixed fertilizer (struvite + DAP) on three crops. Chemical fertilizers, such as DAP, increased P availability by 67–80% more than struvite, depending on the crop. However, struvite increased available P by 100–200% over the control. However, studies show that the slow-release properties of struvite make it as effective as TSP by the end of the trial [4,25]. Vaneeckhaute et al. [47] showed an increase in directly available soluble P from 0% to 75% compared to TSP in about 2 weeks.

In general, the ability of microorganisms to solubilize phosphate via enzymes is briefly described in the literature [1,17]. Inoculation with microorganisms showed a remarkable positive impact on phospho-mono-esterase activity, especially when combined with struvite fertilization. Similar studies have shown an increase in phosphatases in crops inoculated with microorganisms and different types of fertilizer [12,42]. In contrast, AlP exhibited a more pronounced response to this interaction compared to acidic AcP activity. Moreover, the impact of inoculated struvite and TSP on AcP activity was comparable, underlining the efficacy of both fertilizers. In soils with high acidity, the combination of struvite and microorganisms may be particularly effective, as the solubility of struvite in acidic environments enhances the role of AcP in plant growth processes, optimizes soil biochemical processes and facilitates optimal symbiotic interactions for crops [48]. It has been observed that phosphatase is suppressed by inorganic P availability and enhanced by organic P availability. It has also been found that increasing soil P levels also increases phosphatase levels [48].

In this context, microorganisms such as yeast have demonstrated the capacity to enhance enzyme activity, thereby facilitating increased bioavailability of nutrients [12]. Phospho-mono-esterases exhibit pH-dependent activity, with optimal functioning occurring within a specific pH range, which can be either acidic or alkaline. These enzymes act as hydrolases and are capable of solubilizing approximately 90% of the inorganic phosphate (PO₄³⁻) from both organic and inorganic compounds, thereby increasing the bioavailability of P for plant uptake [1]. Yeasts display a range of intriguing PGP characteristics, including the capacity to produce enzymes, solubilize phosphate, interact with soil microbiota, enhance fertilization and, notably, withstand other abiotic stresses [12,17].

The results of the multivariate analysis indicated that both inoculation and fertilization have a complementary effect. Inoculation with N. albida has been shown to reduce oxidative stress and enhance plant growth by improving the bioavailability of key nutrients, such as P and N. This is particularly evident when struvite or TSP is used as fertilizer. These findings suggest that the potential improvement of plant oxidative status may be significantly enhanced by the presence of organisms with multiple PGP capacities, while fertilization with TSP and struvite are related to variables such as chlorophyll content (Chl a, Chl b, Total Chl), nutrients, and enzyme activity (AcP, AlP). Both struvite and TSP were efficient as P sources but differed in the mode of application. TSP is fast-releasing, while struvite is slow-releasing, which could explain its association with long-term growth variables.

Struvite has been shown to have a synergistic effect on plant nutrition, reducing oxidative stress and promoting crop development. These findings highlight the importance of integrating nutrient and microorganism management strategies for a more sustainable agriculture. From a global perspective, the reuse of struvite fits into an institutional framework for P recycling [49]. Environmentally, the application of struvite within a circular economy context helps mitigate the overexploitation of P resources and reduce water pollution, particularly eutrophication and runoff.

This is further supported by the efficient use of P in agriculture, where micro-organisms facilitate P assimilation, reducing losses in different soil types depending on the soil and micro-organisms in each region. The adoption of struvite-based fertilizers can generate significant social benefits with indirect economic value by promoting food self-sufficiency and reducing dependence on external inputs, thereby strengthening food security in vulnerable communities. Consequently, this research provides a scientific foundation for public policy decisions, which should always be accompanied by an integrated approach that includes technological aspects, economic incentives, public acceptance, and scientific data, in order to ensure public health and the conservation of resources [49].

5. Conclusions and Future Perspectives

This study highlights the potential of Naganishia albida as an effective P solubilizing microorganism, outperforming others in struvite solubilization. Its ability to lower pH and release organic acids enhances P availability for plant uptake. Inoculation with N. albida improved lettuce growth and P assimilation compared to traditional fertilizers, like TSP. While antioxidant activity did not differ significantly across fertilizer types, inoculation helped mitigate oxidative stress, promoting plant health. The results of this study provide important evidence on the efficiency of using struvite as a recycled fertilizer and the fundamental role of microorganisms in improving P bioavailability, contributing to more efficient and environmentally friendly agriculture. Future studies should investigate the long-term effects of different P sources, considering the interactions between microorganisms and soil types in different regions. The mechanisms of action of these micro-organisms and their relationship with different environmental conditions need to be further investigated. In addition, future research can focus on life cycle based environmental assessments, contributing to the development of more efficient and adapted biofertilizers and bio-stimulants, thus promoting more sustainable agriculture. This holistic approach will support the transition to circular and resilient agriculture, in line with global sustainability and food security challenges.