Comparative Efficiency of Fungal Organic Acids and Pure Acids in Tricalcium Phosphate Solubilisation

Abstract

Phosphorus (P) is a vital macronutrient involved in key biochemical processes that support plant growth; however, its low bioavailability in agricultural soils remains a major constraint on crop productivity. This limitation is commonly addressed through the application of chemical P fertilisers produced by acidulation of phosphate rock (PR), a process that is costly, energy-intensive, and environmentally hazardous. This study evaluated the P-solubilising potential of culture filtrates from three fungal strains (Aspergillus flavus JKJ7, Talaromyces purpureogenus JKJ12, and Trichoderma koningiopsis JKJ18) grown in National Botanical Research Institute’s Phosphate (NBRIP) liquid medium supplemented with tricalcium phosphate (TCP), and compared their TCP solubilisation efficiency with that of pure acids (citric and sulfuric acid). All three fungal strains solubilised TCP in NBRIP medium, with A. flavus JKJ7 producing the highest concentration of soluble P (259.81 mg L⁻¹), followed by T. koningiopsis JKJ18 (166.41 mg L⁻¹) and T. purpureogenus JKJ12 (47.07 mg L⁻¹). Soluble P concentrations were inversely correlated with pH and positively correlated with titratable organic acidity (TOA). High-performance liquid chromatography (HPLC) identified citric, succinic, tartaric, and gluconic acids as the dominant organic acids associated with P solubilisation. In pure acid treatments, sulfuric acid exhibited concentration-dependent increases in soluble P, whereas citric acid showed reduced solubilisation efficiency at higher concentrations. Although fungal culture filtrates achieved lower maximum TCP solubilisation than strong mineral acids, their higher TOA contributed to improved stabilisation of soluble P by limiting calcium-mediated reprecipitation. These findings demonstrate that crude fungal organic acid mixtures can complement or partially substitute inorganic acids for mobilising P from low-reactivity PR, offering a potentially cost-effective and environmentally sustainable alternative for P fertiliser production. This study supports the development of biologically derived P inputs aligned with circular bioeconomy and sustainable agriculture goals.

1. Introduction

Phosphorus (P) is an essential macronutrient required for plant growth and development due to its central role in energy transfer (ATP), nucleic acid synthesis, and membrane structure. Despite its abundance in many soils, P deficiency remains one of the most widespread constraints to global crop productivity, largely due to the predominance of insoluble P forms that are inaccessible to plants [1,2]. To address this limitation, modern agriculture relies heavily on chemical P fertilisers manufactured from phosphate rock (PR) through acidulation processes that yield water-soluble products such as single superphosphate (SSP) and triple superphosphate (TSP) [3,4]. Although effective, these processes are energy-intensive, costly, and associated with greenhouse gas emissions, heavy metal accumulation, and long-term soil degradation [5,6]. In addition, high-grade PR reserves are non-renewable and are being rapidly depleted, highlighting the urgent need for more sustainable P management strategies.

Direct application of PR has been proposed as a low-cost alternative, particularly in acidic soils where natural dissolution may occur [5]. However, many PR sources, including igneous phosphate rocks such as Dorowa phosphate rock (DPR), exhibit extremely low solubility due to their fluorapatite structure, limiting their agronomic effectiveness across most soil types [3]. This has driven increasing interest in biological approaches to P mobilisation, particularly through the use of phosphate-solubilising microorganisms (PSMs), as environmentally friendly and renewable alternatives for improving P availability.

Phosphate-solubilising microorganisms, including both bacteria and fungi, mobilise insoluble P through several mechanisms, including organic acid secretion, proton extrusion, and enzymatic activity [7,8]. Among these mechanisms, the production of low-molecular-weight organic acids (LMWOAs) is considered the most influential, as acids such as citric, oxalic, and gluconic acids chelate metal cations (Ca²⁺, Fe³⁺, and Al³⁺) that bind phosphate, thereby releasing soluble P into solution [9,10]. Fungi, particularly species belonging to Aspergillus, Talaromyces, and Trichoderma, are recognised as effective P solubilisers due to their high organic acid productivity and extensive hyphal networks, which enhance substrate interaction [11,12].

Despite their potential, the performance of PSMs under field conditions is often inconsistent. Rapid degradation of organic acids, competition with native soil microbiota, and environmental constraints such as pH, temperature, and moisture can significantly limit microbial P solubilisation in situ [13]. To overcome these challenges, ex situ microbial P solubilisation has emerged as an alternative strategy. In this approach, microorganisms are cultured under controlled laboratory conditions to maximise organic acid production, after which their acid-rich culture filtrates are applied directly to PR. This method enables controlled P mobilisation while avoiding uncertainties associated with microbial survival and activity in soil environments [14,15]. Although this strategy has shown promise for upgrading low-reactivity PR, comparative assessments of fungal culture filtrates and their performance relative to pure organic and inorganic acids remain limited.

This study investigated the P-solubilising potential of culture filtrates from three fungal strains, A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningiopsis JKJ18, grown in NBRIP liquid medium supplemented with TCP. These strains were previously shown to possess plant growth-promoting traits, including P solubilisation [16]; however, their organic acid profiles and relative contributions to TCP dissolution have not been systematically compared.

The objectives of this study were to (i) quantify and compare the TCP solubilisation efficiencies of fungal culture filtrates, (ii) characterise the associated LMWOA production profiles, and (iii) evaluate fungal culture filtrate performance relative to pure organic and inorganic acids. It was hypothesised that fungal culture filtrates, owing to their complex mixtures of organic acids and associated metabolites, would exhibit solubilisation behaviours that differ mechanistically from those of pure organic and inorganic acids under controlled dissolution conditions, reflecting differences in acid composition, chelation dynamics, and solution stability rather than absolute dissolution capacity. Among the evaluated strains, A. flavus JKJ7 was expected to display distinct solubilisation characteristics associated with higher organic acid production, based on its previously reported phosphate-solubilising traits. This study treats fungal culture filtrates and pure organic acids as chemically distinct solubilisation systems, without assuming inherent superiority of biologically derived filtrates over defined acid solutions. The findings of this study contribute to the development of biologically derived strategies for mobilising P from low-reactivity PR, supporting more sustainable alternatives to conventional acid-based fertiliser production.

2. Materials and Methods

2.1. Fungal Strains and Inoculum Preparation

Three phosphate-solubilising fungal (PSF) strains, Aspergillus flavus JKJ7, Talaromyces purpureogenus JKJ12, and Trichoderma koningiopsis JKJ18, previously isolated from soils collected from decomposed plant material at the University of Limpopo and a garden site in Polokwane, South Africa, were used in this study [16]. These strains had been previously characterised for phosphate-solubilising potential. Fungal cultures were maintained on potato dextrose agar (PDA) at 4 °C for storage. Prior to experimentation, the strains were subcultured onto fresh PDA plates and incubated at 30 °C for 7 days to obtain actively growing monocultures. These 7-day-old cultures served as inoculum sources for all subsequent experiments.

2.2. Preparation of Fungal Culture Filtrates

Actively growing monocultures of A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningiopsis JKJ18 were inoculated into National Botanical Research Institute’s Phosphate (NBRIP) liquid medium containing 10 g L⁻¹ glucose, 5 g L⁻¹ MgCl₂·6H₂O, 0.25 g L⁻¹ MgSO₄·7H₂O, 0.2 g L⁻¹ KCl, 0.1 g L⁻¹ (NH₄)₂SO₄, and 5 g L⁻¹ tricalcium phosphate (TCP) as the sole phosphorus source [17]. The initial pH of the medium was 6.73. For comparative assessment in a complex nutrient medium, fungal strains were also cultured in potato dextrose broth without TCP supplementation (PDB–TCP). All media were sterilised by autoclaving at 121 °C for 15 min. Fungal inoculation was performed by aseptically transferring a 1 cm diameter mycelial plug from a 7-day-old PDA culture into 250 mL Erlenmeyer flasks containing 100 mL of either NBRIP+TCP or PDB–TCP. All cultures were prepared as independent biological replicates (n = 3 per treatment) and incubated at 30 °C with continuous shaking at 150 rpm for 7 days. The single incubation period was selected to enable a comparative assessment of acid-mediated solubilisation capacity; however, its limitation in capturing temporal dynamics of acid production is acknowledged. Following incubation, fungal biomass was removed by filtration through Whatman No. 1 filter paper (Cytiva, Maidsone, UK). The resulting cell-free culture filtrates were collected and stored at 4 °C prior to further analyses and TCP solubilisation assays.

2.3. Determination of Soluble Phosphorus, pH, and Titratable Organic Acidity

Soluble phosphorus concentrations in fungal culture filtrates were quantified spectrophotometrically using the ascorbic acid method at 820 nm, with KH₂PO₄ used to generate calibration curves [18]. Results are expressed as mg L⁻¹ soluble P. The pH of each filtrate was measured using a calibrated pH meter. Titratable organic acidity (TOA) was determined by titration with 0.1 M NaOH following established protocols [19,20]. Titration was performed gradually, and the endpoint was defined at pH 8.2–8.4. TOA was calculated using the following formula:

TOA (mmol L⁻¹) = (V × M × 1000)/sample volume

where V is the volume of NaOH consumed (L), and M is the molarity of NaOH.

Organic acids present in the fungal culture filtrates were identified using high-performance liquid chromatography (HPLC). Separation was performed on a C18 reverse-phase column (4.6 × 250 mm, 5 µm) at 35 °C under isocratic conditions with a flow rate of 1 mL min⁻¹. The mobile phase consisted of 25 mM K₂HPO₄, with hydrochloric acid (HCl) used to adjust the pH to 2.1. Detection was conducted at 200, 205, and 210 nm, and retention times were compared with standards of ascorbic, citric, fumaric, gluconic, lactic, succinic, and tartaric acids [21]. Organic acid analysis was qualitative in nature. All analyses were conducted in triplicate.

2.4. TCP Solubilisation by Fungal Culture Filtrates and Pure Acids

The phosphorus-solubilising capacity of fungal culture filtrates was evaluated using TCP as a model insoluble phosphorus source. TCP solubilisation assays were conducted by suspending 0.1 g TCP in 10 mL of NBRIP-based fungal culture filtrate in 100 mL DURAN^® Schott bottles (Schott AG, Mainz, Germany) [22]. An uninoculated NBRIP+TCP medium served as the control. Suspensions were agitated at 150 rpm and incubated at 70 °C for 60 min to facilitate dissolution, following a previously described accelerated comparative solubilisation method [23]. This approach was used to enable consistent comparison between biological filtrates and pure acid treatments, while acknowledging that the elevated temperature does not represent natural soil conditions. Following incubation, samples were centrifuged at 10,000 rpm for 10 min, and the supernatants were analysed for soluble phosphorus content using the ascorbic acid method [16,18]. For comparison with chemical solubilisation, parallel TCP dissolution assays were conducted using citric acid and sulfuric acid solutions at concentrations of 5, 10, 25, and 50 mM. Distilled water served as a negative control. All treatments were performed in independent biological triplicates, and results are reported as mean values.

2.5. Statistical Analysis

Statistical analyses were performed using GraphPad Prism version 9.5.0 (GraphPad Software, San Diego, CA, USA). All experiments were conducted in biological triplicate, and data are presented as mean ± standard deviation (SD). Prior to analysis, the data were assessed for normality and homogeneity of variances. A one-way analysis of variance (ANOVA) was used to compare multiple treatments against a single control, followed by Dunnett’s post hoc test for multiple comparisons. Statistical significance was defined at p < 0.05.

3. Results

3.1. Change in Soluble P Levels in Both NBRIP+TCP and PDB-TCP Liquid Media

The three fungal strains exhibited varying capacities to solubilise P from TCP during the seven-day cultivation period in NBRIP+TCP liquid medium. Soluble P levels increased in all inoculated media compared with both their initial concentrations and the uninoculated control (Figure 1). Among the fungal strains, A. flavus JKJ7 demonstrated the highest P solubilisation, reaching 259.81 mg/L, followed by T. koningiopsis JKJ18 at 166.41 mg/L, and T. purpureogenus JKJ12 at 47.07 mg/L. The uninoculated control reached a final soluble P concentration of only 16.54 mg/L. In contrast, cultivation in PDB-TCP resulted in a general decline in soluble P concentrations over the seven days for all fungal strains. The uninoculated PDB-TCP control remained unchanged, indicating that no P solubilisation or utilisation occurred in the absence of fungal activity. These findings demonstrate that the medium composition, particularly the form and availability of P, strongly influences fungal P-solubilising activity.

Figure 1.

The chart represents resultant soluble P culturing three fungal strains (A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningiopsis JKJ18) after culturing the fungal strains in PDB-TCP and NBRIP+TCP liquid media. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. Pairwise comparisons between soluble P in NBRIP fungal cultures and the uninoculated NBRIP control, as well as comparisons between soluble P in PDB fungal cultures and the uninoculated PDB control, are indicated by horizontal lines with significance levels: ** p < 0.01, **** p < 0.0001; ns = not significant.

3.2. Accumulation of TOA and pH Change in the NBRIP+TCP and PDB-TCP Liquid Media

P solubilisation by the fungal strains in NBRIP+TCP liquid medium was accompanied by acidification (Table 1). The fungal strain A. flavus JKJ7 caused the greatest pH reduction, from 6.73 to 4.75, followed by T. koningiopsis JKJ18 (5.65). In contrast, the fungal strain T. purpureogenus JKJ12 slightly increased the pH to 7.52, deviating from the expected acidification trend. In PDB-TCP cultures, the three fungal strains caused a pH increase over the seven days of incubation. The fungal strain T. koningiopsis JKJ18 showed the most pronounced rise, from 5.02 to 7.54, followed by A. flavus JKJ7 (7.24) and T. purpureogenus JKJ12 (5.46). These pH changes in PDB-TCP reflect the nutrient-rich nature of the medium, which likely downregulates acid production as the fungi prioritise carbon metabolism over organic acid secretion. Titration with NaOH revealed that the culture filtrate of A. flavus JKJ7 contained the highest concentration of TOA (12.3 mM), followed by T. koningiopsis JKJ18 (2.2 mM) and T. purpureogenus JKJ12 (0.69 mM) (Figure 2). In PDB-TCP cultures, TOA concentrations decreased across all fungal treatments, consistent with the observed pH increases. The uninoculated PDB-TCP control served as a reference point.

Table 1.

The final pH of the NBRIP+TCP and PDB-TCP fungal culture filtrate from their initial pH, which was equal to their respective controls (A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningiopsis JKJ18).

Liquid Media	JKJ7	JKJ12	JKJ18	Control
NBRIP+TCP	4.75	7.52	5.65	6.73
PDB-TCP	7.24	5.46	7.54	5.02

Figure 2.

The chart represents the TOA content in both PDB-TCP and NBRIP+TCP culture filtrates of the three fungal strains (A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningiopsis JKJ18). Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. Pairwise comparisons between TOA in NBRIP fungal cultures and the uninoculated NBRIP control, as well as comparisons between TOA in PDB fungal cultures and the uninoculated PDB control, are indicated by horizontal lines with significance levels: ** p < 0.01, **** p < 0.0001; ns = not significant.

Table 2 presents the organic acids detected in the NBRIP+TCP culture filtrates of the three fungal strains, as identified by HPLC. Several organic acids known to contribute to P solubilisation were detected. The fungal strain A. flavus JKJ7 produced six of the seven targeted organic acids, whereas both T. purpureogenus JKJ12 and T. koningiopsis JKJ18 produced four, with differences in their acid profiles. Notably, succinic, tartaric, and citric acids were produced by all three fungal strains.

Table 2.

Presence (+) or absence (−) of organic acids in the NBRIP+TCP fungal culture filtrates controls (A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningeopsis JKJ18).

Organic Acids	JKJ7	JKJ12	JKJ18	Control
Lactic acid	+	−	−	−
Succinic acid	+	+	+	−
Fumaric acid	−	+	−	−
Ascorbic acid	+	−	−	−
Gluconic acid	+	−	+	−
Tartaric acid	+	+	+	−
Citric acid	+	+	+	−

3.3. Effects of the NBRIP+TCP Culture Filtrate and Pure Organic and Inorganic Acid Treatment on the Release of P from TCP

When TCP was treated with pure acids, distinct solubilisation patterns were observed (Figure 3). Sulfuric acid treatments resulted in a concentration-dependent increase in soluble P, reflecting its strong protonating and mineral-dissolving capabilities. Conversely, increasing citric acid concentrations led to reduced soluble P levels, likely due to Ca–citrate complex formation, which restricted free P availability. This observation aligns with previous reports that high concentrations of organic acids can induce P precipitation by complexing with cations such as Ca²⁺.

Figure 3.

Comparative analysis of the TCP solubilisation efficiency between the citric (organic) and sulfuric (inorganic) acid treatments at different concentrations, with water serving as a control. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. Pairwise comparisons between the soluble P caused by the acid treatments and water control are indicated by horizontal lines with significance levels: **** p < 0.0001; ns = not significant.

Interestingly, TCP solubilisation using the fungal culture filtrates showed a slight decrease in soluble P concentration following treatment, although the extent of reduction varied among strains. The filtrate from A. flavus JKJ7, which exhibited the highest TOA and most diverse acid profile, retained the greatest proportion of soluble P. This suggests a stabilising effect of mixed organic acids that prevents the rapid reprecipitation of P, likely through sustained chelation of calcium ions and buffering of the solution pH. In contrast, filtrates from T. koningiopsis JKJ18 and T. purpureogenus JKJ12 exhibited greater decreases in soluble P content, possibly due to less effective acid buffering or lower chelating potential.

Although the fungal filtrates did not surpass strong mineral acids in total solubilisation capacity, their higher titratable acidity and balanced organic acid composition provided more stable P retention. This indicates that fungal-derived organic acids could serve as natural solubilising agents capable of mobilising P from low-reactivity phosphate sources, offering a sustainable and environmentally friendly alternative to conventional acid-based fertiliser production.

The NBRIP+TCP fungal culture filtrates exhibited distinct differences in their ability to dissolve TCP (Figure 4). As shown in Figure 4A, the concentration of soluble P initially present in each culture filtrate was compared with the concentration measured after treatment with TCP, and the net change in soluble P was calculated. A general decrease in the soluble P concentration was observed across all treatments following the TCP solubilisation experiment, with the extent of this reduction presented in Figure 4B. Among the treatments, the culture filtrate of A. flavus JKJ7 showed the smallest decline in soluble P (−1.56 mg/L), followed by T. purpureogenus JKJ12 (−44.76 mg/L) and then T. koningiopsis JKJ18 (−54.32 mg/L). Interestingly, there was no significant difference in soluble P reduction between the uninoculated NBRIP+TCP liquid medium, used as the control, and the A. flavus JKJ7 treatment, highlighting the relative stability and solubilisation efficiency of the latter.

Figure 4.

The effect of NBRIP+TCP culture filtrates of the three PSF (A. flavus JKJ7, T. purpureogenus JKJ12, and T. koningiopsis JKJ18) on TCP dissolution, with (A) comparing the initial soluble P in the culture filtrates and the final concentrations of soluble P after the TCP dissolution experiment, while (B) represents the change in soluble P concentrations after the dissolution experiment. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test for multiple comparisons. Pairwise comparisons between initial and final soluble P before and after treating TCP with NBRIP fungal cultures or the uninoculated NBRIP control (A), as well as comparison between the change in soluble P concentrations after the TCP dissolution experiment by each NBRIP fungal culture filtrate and the uninoculated control (B), are indicated by horizontal lines with significance levels: * p < 0.05, ** p < 0.01; ns = not significant.

4. Discussion

The findings of this study provide mechanistic insight into fungal-mediated phosphorus (P) solubilisation and highlight the contrasting behaviours of biological and chemical solubilisation pathways under controlled laboratory conditions. All three phosphate-solubilising fungal (PSF) strains—Aspergillus flavus JKJ7, Talaromyces purpureogenus JKJ12, and Trichoderma koningiopsis JKJ18—demonstrated the ability to mobilise P from tricalcium phosphate (TCP) when cultured in NBRIP medium, confirming their functional capacity for inorganic P activation.

Consistent with established models of microbial P solubilisation, increases in soluble P concentrations were accompanied by significant reductions in pH and elevated titratable organic acidity (TOA), indicating acid-mediated dissolution mechanisms [10,24,25]. The strong inverse relationship observed between pH and soluble P supports the central role of low-molecular-weight organic acids (LMWOAs) in chelating Ca²⁺ ions and destabilising Ca–P mineral complexes. Organic acids such as citric, gluconic, and oxalic acids have been widely reported as key drivers of fungal P solubilisation through both proton-mediated dissolution and metal chelation pathways [24,26,27].

Among the evaluated strains, A. flavus JKJ7 exhibited the highest acidification capacity and the greatest P solubilisation, suggesting a more efficient redirection of carbon flux toward organic acid synthesis. This observation aligns with reports describing high strain-level variability among Aspergillus species, with certain isolates achieving exceptionally high soluble P concentrations under optimised conditions [28,29,30]. Previous studies have also linked strong organic acid production in A. flavus to enhanced plant growth promotion and metal mobilisation, indicating that the in vitro solubilisation capacity observed here may translate into agronomically relevant functionality under appropriate deployment conditions [28].

The absence of measurable P solubilisation in PDB–TCP medium further highlights the regulatory nature of fungal acid production. In nutrient-rich environments where phosphorus and carbon are readily available, fungi appear to downregulate energetically costly organic acid synthesis pathways [31,32]. This substrate-dependent response underscores the importance of C:P ratio, P limitation, and medium composition in governing microbial solubilisation behaviour [10,33]. Such findings are particularly relevant for the design of biological solubilisation systems, as they indicate that effective P mobilisation is contingent on maintaining metabolic conditions that favour acid excretion rather than biomass accumulation.

Fungal organic acid production is a tightly regulated metabolic process driven by nutrient stress and environmental cues. Under P-deficient conditions, fungi reroute central carbon metabolism toward the synthesis and excretion of carboxylic acids, often coupled with proton extrusion via plasma membrane ATPases [34,35,36]. These acids promote P mobilisation primarily through chelation, whereby carboxyl functional groups bind Ca²⁺, Fe³⁺, or Al³⁺ ions that otherwise immobilise phosphate in mineral matrices [26,27]. The diversity and relative proportions of these acids are therefore critical determinants of solubilisation efficiency and stability.

Comparison with pure acid treatments revealed important distinctions between chemical and biological solubilisation mechanisms. Sulfuric acid displayed a clear concentration-dependent increase in soluble P, reflecting its strong proton-donating capacity and non-selective mineral dissolution behaviour. Citric acid exhibited a more complex response, with lower concentrations enhancing P release, while higher concentrations reduced soluble P, likely due to the formation of Ca–citrate complexes that limit free phosphate availability [24]. Such dual behaviour of organic acids has been previously reported and reflects the balance between proton activity and chelation-driven precipitation processes [10].

Contrary to the initial hypothesis, pure acids outperformed fungal NBRIP culture filtrates in terms of maximum TCP solubilisation. However, this outcome does not diminish the functional relevance of the fungal filtrates. Treatment of TCP with fungal culture filtrates resulted in a net reduction in soluble P relative to the uninoculated control, with the magnitude of reduction varying among the strains. Notably, the A. flavus JKJ7 filtrate, which exhibited the highest TOA, showed the smallest decline in soluble P following TCP treatment. This suggests that complex mixtures of fungal-derived organic acids may contribute to the stabilisation of soluble P by maintaining Ca²⁺ in chelated forms or buffering solution pH, thereby limiting rapid reprecipitation of Ca–P phases. Similar stabilisation effects have been reported for Aspergillus niger, where mixed organic acid systems reduced Ca–P recrystallisation despite lower peak solubilisation [24,37].

Strain-specific differences observed among T. koningiopsis JKJ18 and T. purpureogenus JKJ12 further indicate that organic acid composition, rather than total acidity alone, governs solubilisation outcomes. The potential contribution of additional extracellular metabolites, including siderophores or phosphatases, may also influence P mobilisation and warrants further investigation [38,39].

The elevated temperature (70 °C) employed in the TCP dissolution assays was used as an accelerated comparative approach to enhance reaction kinetics and to allow for clear differentiation among solubilisation treatments within a constrained experimental timeframe. Increased temperature is known to enhance molecular mobility and diffusion rates, which can accelerate mineral dissolution and transformation processes by lowering activation energy barriers [40]. However, it is important to note that such conditions do not reflect natural soil environments and may significantly alter both the magnitude and dynamics of acid–mineral interactions. At elevated temperatures, the dissolution of calcium phosphates may be accompanied by enhanced secondary processes, including supersaturation-driven reprecipitation of calcium phosphate phases upon cooling or as solution chemistry evolves, and phase transformations that are not representative of ambient soil conditions—phenomena documented in controlled precipitation studies where temperature impacted calcium phosphate transformations and phase stability [40].

Accordingly, the 70 °C assay should be regarded as an accelerated comparative method rather than a soil-simulation model, and the resulting dissolution patterns should be interpreted in a relative context across treatments rather than as direct predictors of in situ soil phosphorus dynamics.

Accordingly, the TCP dissolution data presented here should be interpreted as comparative indicators of solubilisation behaviour across treatments, rather than direct quantitative proxies for soil P mobilisation efficiency under field conditions.

Ionic strength effects further complicate the interpretation of solubilisation outcomes, particularly in systems containing complex mixtures of organic acids. Elevated background electrolyte concentrations influence activity coefficients, suppress effective proton activity, and may reduce ligand chelation efficiency, thereby altering observed phosphate solubility and precipitation–dissolution equilibria [41]. Moreover, specific interactions between organic ligands such as citrate and calcium ions have been shown to non-linearly affect orthophosphate solubility at varying ionic strengths, highlighting the importance of electrostatic and complexation effects in multi-component systems [42].

The fungal culture filtrates used in this study represent chemically complex systems containing multiple organic acids and additional extracellular metabolites. Under accelerated dissolution conditions, these components may engage in competing processes, including Ca²⁺ chelation, pH buffering, and solution stability effects. While strong mineral acids drive rapid TCP dissolution, mixed organic acid systems may reduce peak soluble P concentrations due to ligand interactions and potential reprecipitation control, but concurrently enhance the persistence of soluble phosphate by limiting rapid Ca–P recrystallisation. Such stabilisation behaviour has been observed in mixed organic acid systems where citrate and related anions influence calcium phosphate equilibria by forming stable complexes that prolong supersaturation and delay precipitation, relative to inorganic acidity alone.

Thus, the reduced soluble P observed following treatment with fungal culture filtrates under the conditions of this study should not be interpreted as diminished functional potential per se, but rather as reflective of the distinct chemical equilibria governing biological versus purely mineral acid-driven solubilisation systems. These findings highlight the need to consider both dissolution magnitude and the temporal stability of solubilised P when evaluating biological P-mobilisation strategies.

Overall, these findings indicate that fungal culture filtrates function differently from strong mineral acids, prioritising stabilisation and controlled mobilisation of P rather than rapid dissolution. Optimisation of fungal fermentation conditions, including incubation duration, carbon limitation, and nutrient stress, may further enhance organic acid diversity and solubilisation performance [43]. Importantly, soil-based and field-level validation remains essential to determine whether the stabilisation effects observed in vitro translate into improved plant P uptake and reduced fertiliser losses. Previous studies have demonstrated that Trichoderma spp. and other PSF enhance crop productivity under field conditions, supporting the potential application of fungal-derived solubilisation strategies in sustainable agriculture [44,45]. Integration of fungal organic acids with low-reactivity phosphate sources could contribute to reduced chemical fertiliser dependence and improved nutrient use efficiency, aligning with circular economy and climate-smart agriculture objectives [31,46].

Abbreviations

The following abbreviations are used in this manuscript:

HPLC	High-Performance Liquid Chromatography
NBRIP	National Botanical Research Institute’s Phosphate
PDA	Potato Dextrose Agar
PDB	Potato Dextrose Broth
PSF	Phosphate-Solubilising Fungi
PSMs	Phosphate-Solubilising Microorganisms
TCP	Tricalcium Phosphate
TOA	Titratable Organic Acidity
PR	Phosphate Rock

Author Contributions

Conceptualisation, N.N.P. and T.J.M.; methodology, T.J.M.; validation, N.N.P. and L.M.; formal analysis, T.J.M.; investigation, T.J.M.; resources, N.N.P.; data curation, T.J.M. and N.N.P.; writing—original draft preparation, T.J.M.; writing—review and editing, T.J.M., N.N.P., L.M., and E.L.J.V.R.; supervision, N.N.P., L.M., and E.L.J.V.R.; project administration, N.N.P.; funding acquisition, N.N.P. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.