Optimization of phosphate solubilization by Pseudomonas salmasensis using response surface methodology

Otimização da solubilização de fosfato por Pseudomonas salmasensis usando metodologia de superfície de resposta

Abstract

This study aimed to analyze the effects of incubation temperature and contents of glucose and phosphate rock powder on pH, acidity, and P solubilization by Pseudomonas salmasensis. Nineteen experiments were defined using response surface methodology based on a central composite design (CCD), and the effects of varying incubation temperature, glucose, and phosphate rock powder (coded as −1, 0, or +1) on pH, acidity, and P solubilization were explored. The results revealed that the CCD model had high efficiency in predicting pH, acidity and P solubilization (R² = 0.9421, 0.9240 and 0.9348, respectively). The parameters that exerted the greatest percentage effect on the dependent variables were glucose (26.14%), temperature squared (27.52%) and the interaction between glucose and phosphate rock powder (22.01%) for the pH; temperature (19.82%) for acidity and temperature (34.01%) and rock powder for phosphate solubilization. The optimal operating parameters for maximum phosphate solubilization (57.73 mg L^-1) corresponded to: 28.35 °C, 64.93 g L^-1 of glucose and 2.61 g of natural phosphate. Under these same conditions, the pH and titratable acidity in the culture medium were 4.31 and 213.77 mg L^-1, respectively. The ANOVA analysis confirmed the accuracy and validity of the model in terms of F value (1990.93 and 207.16 for acidity and phosphorus, respectively), all significant with p < 0.001.

Keywords:
phosphate-solubilizing bacteria; central composite planning; nutrient

Resumo

Objetivou-se com este estudo analisar o efeito da temperatura de incubação e de teores de glicose e de pó de rocha fosfática sobre o pH, acidez e solubilização de P porPseudomonas salmasensis. Dezenove experimentos foram definidos pela metodologia de superfície de resposta com base em um delineamento composto central (DCC), e os efeitos da variação da temperatura de incubação, glicose e pó de rocha fosfática (codificados por −1, 0 ou +1) no pH, acidez e solubilização de P foram explorados. Os resultados revelaram que o modelo DCC teve alta eficiência para prever o pH, a acidez e a solubilização de P (R²= 0,9421; 0,9240 e 0,9348, respectivamente). Os parâmetros que exerceram o maior efeito percentual sobre as variáveis dependentes foram: glicose (26,14%), temperatura ao quadrado (27,52%) e a interação entre glicose e pó de rocha (22,01%) para o pH; temperatura (19,82%) para acidez e temperatura (34,01%) e pó de rocha para solubilização de fosfato. Os parâmetros operacionais ótimos para a solubilização máxima de fosfato (57,73 mg L^-1) corresponderam a: 28,35 °C, 64,93 g L^-1 de glicose e 2,61 g de fosfato natural. Nestas mesmas condições, o pH e a acidez titulável no meio de cultura apresentaram os seguintes valores: 4,31 e 213,77 mg L^-1, respectivamente. A análise ANOVA confirmou a precisão e validade do modelo em termos de valor F (0,26; 1990,93 e 207,16) para pH, acidez e teor de fósforo, respectivamente, todos significativos com valor p < 0,001.

Palavras-chave:
bactéria solubilizadora de fosfato; planejamento composto central; nutriente

1. Introduction

The availability of phosphorus (P) is essential for plant growth and development in tropical soils. However, its limited bioavailability poses a significant challenge to agricultural productivity, since in acidic soils this element is fixed by reactions that result in the formation of insoluble precipitates with aluminum (Al) and iron (Fe) oxides (Johan et al., 2021), while under alkaline conditions, low-solubility calcium phosphates are formed (Khan et al., 2023).

In the Brazilian context, most soils exhibit a high degree of weathering, a characteristic that intensifies phosphate retention and restricts its availability for economically important crops such as corn and sugarcane (Dias et al., 2022; Oliveira et al., 2022). As a consequence, the agronomic efficiency of soluble phosphate fertilizers tends to be reduced, requiring applications in high doses to meet the demands (Zhou et al., 2022).

Among the most prominent mechanisms, the ability to solubilize insoluble phosphates from soil amendments and alternative phosphorus sources, such as phosphate rock, stands out, as reported by Amri et al. (2022). These authors demonstrated that co-inoculation with Pseudomonas fluorescens promoted greater root proliferation, increased biomass, and enhanced phosphorus uptake in ryegrass, outperforming conventional phosphate fertilizers while maintaining adequate soil nutrient levels at a lower cost. Similarly, Vey et al. (2025) reported that inoculation with the same bacterial species increased leaf phosphorus concentration and soybean root dry biomass when phosphorus levels in the nutrient solution were increased, further highlighting the potential of Pseudomonas spp. to improve phosphorus acquisition and plant growth under enhanced P availability.

In this context, phosphate-solubilizing microorganisms are consolidating as a promising strategy to increase the efficiency of P use in agriculture (Ghoreshizadeh et al., 2024; Al-Tammar and Khalifa, 2022). As described by Xie et al. (2021), the production of organic acids, especially gluconic acid, by Pseudomonas species constitutes a central mechanism in the release of P retained in minerals, increasing its bioavailability in the soil. However, the efficiency of this process is not constant, being strongly influenced by environmental and nutritional variables, such as carbon availability (e.g., glucose), the nature and concentration of the phosphate source (such as phosphate rock), and temperature. These factors directly influence bacterial metabolic activity and solubilization reactions, highlighting the need for optimization strategies that maximize solubilizing performance under controlled conditions.

Although previous studies have evaluated these factors separately, the simultaneous interaction between glucose, rock powder, and temperature on pH, acidity, and P solubilization is still poorly understood. However, increased glucose is expected to stimulate microbial activity, reducing pH and favoring P solubilization (Sanchez-Gonzalez et al., 2023). Phosphate rock may also act as a pH-buffering mineral source, depending on its composition (Marcotte et al., 2020), while temperature can optimize or limit both biochemical reactions and mineral dissolution rates (Luo et al., 2024; Sharma et al., 2024).

In this context, this study aimed to optimize phosphate solubilization by Pseudomonas salmasensis and evaluate the interactive effects of incubation temperature, glucose content, and phosphate rock powder on pH, acidity, and phosphorus content. Response surface models were used to identify the optimal conditions that maximize phosphorus solubilization.

2. Materials and Methods

2.1. Bacterial isolate and inoculum preparation

Pseudomonas salmasensis CFB 004 isolate was obtained from the rhizosphere of potato plants (Solanum tuberosum) grown in the Mandala garden under agroecological management, without the use of chemical fertilizers, at the Universidade Estadual da Paraíba (UEPB), Campus II, Lagoa Seca, Paraíba. Pseudomonas salmasensis was identified through sequencing of the 16S rRNA and rpoB genes, showing 99% identity to reference sequences deposited in GenBank. The isolate's sequence was deposited in GenBank under accession number PZ159344.1). The isolate was preserved in tubes containing Nutrient Agar (NA) and covered with mineral oil at 4 °C at the Phytopathology Laboratory.

The bacterial isolate was cultured on Petri dishes containing NA for 48 h at 25°C in a BOD incubator in the dark. After incubation, the bacterial cells were suspended in saline solution (0.85% NaCl), and the suspension was adjusted to an optical density of 1.0 at 600 nm. Subsequently, serial dilution and plating on NA medium were performed to determine cell concentration. Then, 10 µL of the bacterial suspension (3.5 x 10¹⁰ CFU mL⁻¹) was added at four equidistant points on a Petri dish (9 cm in diameter) containing Pikovskaya's Agar medium. The plates were kept in a BOD incubator in the dark at 25°C for 10 days.

2.2. Qualitative phosphate solubilization (PSI)

Bacterial solubilizing activity was assessed by daily measurement of the solubilization halo formed around growing colonies for seven days. Equation 1 was used to calculate the Phosphate Solubilization Index (PSI) (Waday et al., 2022):

$PSI=\frac{Totaldiameter\left(colony+halozone\right)}{Colonydiameter}$ (1)

Ten Petri dishes, each containing four bacterial colonies, were used to obtain the mean PSI. Thus, the final value was calculated based on 40 independent experimental units.

2.3. Quantitative phosphate solubilization in liquid médium

After determining the phosphorus solubilization index, a quantitative assessment of this nutrient's solubilization was conducted using the Pseudomonas salmasensis CFB 004 isolate in liquid NBRIP medium (10 g L⁻¹ glucose, 0.15 g L⁻¹ (NH₄)₂SO₄, 0.2 g L^-1 KCl, 5 g L^-1 MgCl₂.6H₂O and 0.25 g L^-1 MgSO₄.7H₂O), as described by Nautiyal (1999), without the addition of agar. The medium was supplemented with 5 g L^-1 of commercial Maxgreen® rock poder composed predominantly of apatite [Ca₁₀(PO₄)₆(F,Cl,OH)₂] and containing 29% total phosphorus (P₂O₅), 14% citric acid-soluble phosphorus, and 32% total calcium with a particle size of less than 100 mesh.

Prior to sterilization, the pH of the NBRIP medium was adjusted to 7.0 by adding NaOH and HCl solutions. The experiment was conducted using a completely randomized design with 30 replicates per treatment. The treatments consisted of: T1 = 50 mL of sterile distilled water supplemented with rock powder; T2 = 50 mL of NBRIP medium supplemented with rock powder; and T3 = 49 mL of NBRIP medium supplemented with rock powder and inoculated with 1 mL of the bacterial suspension, resulting in a final concentration of 1 × 10⁶ CFU mL^-1. The cell concentration of the inoculum was previously determined via serial dilutions and plating on Nutrient Agar (NA) medium, as described earlier.

Each experimental unit consisted of a 250 mL Erlenmeyer flask containing the respective treatments. All materials and culture media were autoclaved at 121 °C for 20 min. After cooling to room temperature, inoculation was performed in a laminar flow hood using the bacterial isolate (CFB 004), adjusted to a final concentration of 1 × 10⁶ CFU mL^-1. Subsequently, the flasks were sealed with paper-covered cotton plugs and incubated for 10 days on an orbital shaker (Nova Técnica®) at a constant agitation speed of 55 rpm and a temperature of 25 °C.

2.4. Experimental design for optimization of phosphate solubilization

Based on the confirmation of the phosphate-solubilizing capability of Pseudomonas salmasensis CFB 004 in preliminary qualitative and quantitative assays, an optimization step was conducted to identify the experimental conditions that maximize phosphorus solubilization. To this end, response surface methodology was applied to evaluate the combined effects of temperature, carbon source concentration, and rock phosphate dosage.

The relationships between the different parameters were evaluated using the response surface statistical method with Minitab 18 software. This study employed response surface methodology and a central composite design (CCD), considering the following independent variables: incubation temperature (T), glucose concentration (S), and phosphate rock powder concentration (R). The ranges for temperature, glucose concentration, and rock powder levels used in the experimental design were established based on preliminary phosphorus solubilization assays. The central composite design comprised eight factorial points, six axial points, and five central points, totaling 19 experiments, as shown in Equation 2.

$N=2n+2*n+nc=23+2*3+5=19$ (2)

Where

N - is the total number of experiments required;

n - is the number of variables, and,

nc - is the number of replicates.

The independent variables were set at five coded levels (-α, -1, 0, +1, and +α). The relationship between the coded and actual values ​​of the variables is presented in Table 1.

Table 1
Independent variables and levels used in the central composite design (CCD) for phosphate solubilization and acidity.

Independent Variables	Unit	Coded Symbol	Coded Levels
			-α	-1	0	+1	+ α
Incubation Temperature (T)	°C	T	25.61	28.0	31.5	35.0	37.39
Glucose (S)	g L-1	S	6.07	18.0	35.5	53.0	64.93
Rock Powder (R)	g	R	2.61	5.0	8.5	12.0	14.39

In the experimental design, soluble phosphorus concentration (mg mL⁻¹) was adopted as the response variable. Additionally, pH and acidity were determined at the end of the incubation period. The liquid culture medium used was NBRIP (Nautiyal, 1999), with glucose and phosphate rock powder concentrations adjusted according to the established experimental levels (Table 1). The rock powder used was a commercial product from the Maxgreen® brand. Prior to sterilization, the pH of the NBRIP medium was adjusted to 7.0 using NaOH and HCl solutions. The combination of independent variables and their respective levels, as well as the analyzed variables, is presented in Table 2.

Table 2
Results of the experimental design matrix for the variables analyzed.

Experiment	Coded Variables			Real Variables			Analyzed Variables
	T	S	R	T (°C)	S (g)	R (g L-1)	pH	Acidity mg L-1	P mg L-1
1	-1.00	-1.00	-1.00	28.00	18.00	5.00	6.00	140.89	35.79
2	1.00	-1.00	-1.00	35.00	18.00	5.00	5.30	134.48	16.57
3	-1.00	1.00	-1.00	28.00	53.00	5.00	4.82	185.72	37.77
4	1.00	1.00	-1.00	35.00	53.00	5.00	4.72	115.27	27.88
5	-1.00	-1.00	1.00	28.00	18.00	12.00	4.90	131.00	21.66
6	1.00	-1.00	1.00	35.00	18.00	12.00	5.00	122.00	8.37
7	-1.00	1.00	1.00	28.00	53.00	12.00	4.80	108.87	22.30
8	1.00	1.00	1.00	35.00	53.00	12.00	5.00	51.23	15.30
9	-1.68	0.00	0.00	25.61	35.50	8.50	5.36	121.68	25.61
10	1.68	0.00	0.00	37.39	35.50	8.50	5.30	70.44	1.18
11	0.00	-1.68	0.00	31.50	6.07	8.50	5.10	134.40	20.00
12	0.00	1.68	0.00	31.50	64.93	8.50	4.37	70.00	43.20
13	0.00	0.00	-1.68	31.50	35.50	2.61	5.00	172.91	40.60
14	0.00	0.00	1.68	31.50	35.50	14.39	4.50	153.70	19.10
15	0.00	0.00	0.00	31.50	35.50	8.50	4.70	131.28	22.79
16	0.00	0.00	0.00	31.50	35.50	8.50	4.80	115.27	21.37
17	0.00	0.00	0.00	31.50	35.50	8.50	4.70	134.48	23.63
18	0.00	0.00	0.00	31.50	35.50	8.50	4.70	134.48	21.37
19	0.00	0.00	0.00	31.50	35.50	8.50	4.70	124.88	23.63

• T - Incubation temperature; S - Glucose and R - Rock powder.

For each experimental condition, a 125 mL Erlenmeyer flask containing 50 mL of NBRIP medium was used. The sterilization procedures for materials and culture media, as well as the inoculation of P. salmasensis (CFB 004) at a final concentration of 1 × 10⁶ CFU mL⁻¹, were carried out as described in section 2.3. After inoculation, the flasks were sealed with cotton plugs and incubated for 10 days under static conditions without agitation or forced aeration at the temperatures established for each experimental treatment.

At the end of the incubation period, the liquid fraction obtained from the Erlenmeyer flasks, containing the culture medium and microbial metabolites, was used for the analysis of soluble phosphorus, pH, and acidity.

The relationship between the independent variables and the responses was established using a second-order quadratic polynomial regression model (Equation 3).

$y=\beta 0+{{\displaystyle \sum }}_{i=1}^k\beta ixi+{{\displaystyle \sum }}_{i=1}^k\beta ii{x}_i^2+{{\displaystyle \sum }}_{i=1}^{k-1}{{\displaystyle \sum }}_{j=i+1}^k\beta ij{x}_i{x}_j+\epsilon i\ne j$ (3)

where:

Y - is the response variable (pH, acidity, and solubilized P),

Xi and Xj - are the coded independent variables,

k - is the number of independent variables, ε are the model residuals (difference between observed values ​​and values ​​estimated by the model), and; β0, βi, βii and βij - are the effects of the y-intercept, linear functions, quadratic function, and interaction of the variables, respectively.

To facilitate the interpretation of the modeling results and the classification of the effects of the parameters included in the central composite design model, the percentage contribution of each parameter was estimated via Pareto analysis using Equation 4:

$Pi=\left(\frac{{\beta }_i^2}{\sum {\beta }_i^2}\right)x100i\ne 0$ (4)

Where:

Pi is the percentage effect of each variable included in the CCD model, and

βi represent the coefficient of the polynomial equation.

2.5. Determination of soluble phosphorus, pH, and titratable acidity

For both experimental stages "Quantitative phosphate solubilization in liquid medium" and "Experimental design for the optimization of phosphate solubilization" the response variables (soluble phosphorus, pH, and total titratable acidity) were determined using the same analytical procedures. To this end, at the end of the incubation period, liquid extracts were obtained by gravity filtration of the flask contents using blue band quantitative filter paper.

The soluble phosphorus concentration in the filtered extracts was determined according to the methodology described by Teixeira et al. (2017). A 0.1 mL aliquot of the filtrate was transferred to a 125 mL Erlenmeyer flask, to which 10 mL of a diluted ammonium molybdate solution and approximately 30 mg of powdered ascorbic acid (used as a reducing agent) were added. The samples were agitated on a horizontal circular shaker for 1 to 2 minutes and allowed to stand for 1 hour for blue color development. Subsequently, absorbance was measured using a UV-Vis spectrophotometer at 660 nm, and the soluble phosphorus concentration was determined using a standard curve, as described by Waday et al. (2022).

The total titratable acidity of the filtered extracts was determined according to the procedures described in Standard Methods for the Examination of Water and Wastewater (APHA, 1998a). A 10 mL aliquot of the extract was titrated following the addition of 0.2 mL of phenolphthalein indicator solution. Titration was performed using a 0.02 N NaOH solution previously standardized against potassium hydrogen phthalate (KHC₈H₄O₄) until the color changed from colorless to a persistent pink. Results for total titratable acidity were expressed in citric acid equivalents and calculated using Equation 5:

$Acidity,mg{H}_3{C}_6{H}_5{O}_7{L}^{-1}=\frac{AxB}{mLofsample}\times 64040$ (5)

Where:

A - mL of NaOH titrant consumed in the titration; and,

B - NaOH normality.

The pH of the filtered extracts was determined potentiometrically using a previously calibrated pH meter, with readings taken directly from the samples at the end of the incubation period, as described by APHA, AWWA, and WEF (APHA, 1998b).

Data regarding the response variables (soluble phosphorus, pH, and total titratable acidity) were checked for the assumptions of residual normality and homogeneity of variance. The results indicated that the data met these assumptions, allowing for the application of the statistical methods used in the study.

3. Results

Bacterial growth on Pikovskaya's Agar medium and the solubilization of the inorganic phosphate present in the medium were observed through the formation of a translucent halo around the colony (Figure 1). From a qualitative perspective, the P. salmasensis CFB 004 isolate exhibited an early solubilization profile since the formation of a halo around the colony became evident by the third day of incubation. The calculated Phosphate Solubilization Index was, on average, 2.72, indicating, according to the classification proposed by Chagas Júnior et al. (2010), an intermediate solubilization capacity (2 < PSI < 4).

Figure 1
Formation of translucid halo around colonies of Pseudomonas salmasensis CFB 004 on Pikovskaya's Agar medium, indicating phosphate solubilizing activity.

The maximum available phosphorus was recorded in the sample treated with Pseudomonas salmasensis (Figure 2A), with a value of 77.84 mg L^-1, statistically higher compared to the averages obtained in the other treatments. This result is equivalent to a 5.37% reduction in total P₂O₅ supplied by the rock powder.

Figure 2
Phosphorus (A), pH (B), and acidity (C) levels as a function of the treatments.

A significant reduction in the pH of the medium was observed during the solubilization process (Figure 2B), with the lowest value (4.26) in the inoculated treatment, representing a 40.3% reduction compared to the control treatments (water + rock powder and medium + rock powder). In addition, an increase in titratable acidity was observed in the treatment with the bacteria (Figure 2C).

The ANOVA results showed a significant fit (p < 0.01) to the quadratic regression model for pH, acidity, and soluble phosphorus within the range of the variables studied (Table 3). Most of the model terms significantly influenced the response variable, with the exception of T, S², and R² for pH, T × R for acidity, and the interactions T × S, T × R, and S × R for soluble phosphorus.

Table 3
Analysis of variance for the quadratic response surface model for pH. acidity. and soluble phosphorus content.

Source of variation	DF	pH		Acidity		Phosphorus
		MS	P-value	MS	P-value	MS	P-value
Model	9	0.26	0.00	1990.93	0.00	207.16	0.00
Linear	3	0.34	0.00	2973.29	0.00	470.20	0.00
T	1	0.03	0.14	3861.59	0.00	599.70	0.00
S	1	0.70	0.00	2257.65	0.01	262.60	0.00
R	1	0.29	0.00	2800.63	0.00	548.40	0.00
Quadratic	3	0.26	0.00	1880.92	0.00	136.50	0.00
T × T	1	0.79	0.00	1518.93	0.01	181.00	0.00
S × S	1	0.01	0.30	958.09	0.04	106.50	0.01
R × R	1	0.02	0.22	2387.41	0.00	64.60	0.03
Two-factor interaction	3	0.19	0.00	1118.59	0.01	14.80	0.26
T × S	1	0.06	0.04	1586.99	0.01	30.50	0.10
T × R	1	0.15	0.00	13.04	0.78	9.80	0.33
S × R	1	0.34	0.00	1755.76	0.01	4.10	0.52
Error	9	0.01		162.21		9.20
Lack of fit	5	0.02	0.03	238.66	0.12	15.50	0.02
Pure error	4	0.002		66.64		1.3

• DF - Degrees of freedom; MS - mean squares.

Second-order polynomial equations, adjusted using the response surface methodology, allowed us to describe with high precision the relationship between the experimental factors: incubation temperature (T), glucose concentration (S), and phosphate rock powder content (R), and the response variables (pH, titratable acidity, and soluble phosphorus). Exclusion of statistically insignificant terms resulted in robust predictive models (Equations 6, 7, and 8), whose coefficients indicate the direction and magnitude of the linear, quadratic, and interactive effects.

$pH=4.7685-0.2261S-0.145R+0.232T^2+0.0875TS+0.1375TR+0.2075SR$ (6)

$Acidity=127.94-16.82T-12.86S-14.32R-10.55T^2-8.38S^2+13.22R^2-14.08TS-14.81SR$ (7)

$SolubleP=22.63-6.626T+4.38S-6.337R-3.642T^2+2.793S^2+2.175R^2$ (8)

The regression models (Equations 6, 7, and 8) were highly significant and showed high coefficients of determination (R²) of 94.21, 92.40, and 93.48 for the variables pH, acidity, and solubilized phosphorus, respectively, indicating excellent model fit. Furthermore, the adjusted R² values ​​for these variables, i.e, 91.31, 86.32, and 90.22, respectively, also confirm that the models were highly significant, indicating good agreement between the experimental and predicted values ​​of the dependent variables. The coefficient of variation (CV%) and standard deviation of the three response variables were reasonably low and acceptable, demonstrating a degree of precision and high reliability of the experiments conducted (Table 4).

Table 4
Regression coefficients of the predicted second-order model for the variables pH, acidity, and phosphate solubilization.

Response parameter	pH	Acidity	Solubilized phosphorus
Mean	4.82	123.84	23.59
Standard Deviation	0.109	12.136	3.253
CV%	2.26	9.80	13.79
R2​	94.21	92.40	93.48
Adjusted R2	91.31	86.32	90.22
Suggested Model	Quadratic	Quadratic	Quadratic

• CV- Coefficient of variation; R² - coefficient of determination.

The percentage effect of each parameter obtained by Pareto analysis of the central composite design model for pH, acidity, and P solubilization is shown in Figure 2. It can be seen that the quadratic effect of temperature (27.52%) and the linear effect of glucose (26.14%) promoted the greatest pH variation, the S x R interaction accounted for 22.01%, and the linear effect of rock powder accounted for 10.75% (Figure 2A). As for acidity, the linear effect of temperature promoted the highest percentage contribution (19.82%), the lowest percentages were observed for the quadratic effects of temperature (T2 = 7.80%) and glucose (S2 = 4.92%). while the other parameters. i.e., S, R, R², T x S and S x R, contributed similarly, with percentages ranging from 11.59 to 15.37% (Figure 2B). As for solubilized phosphorus, the linear effect of temperature (34.01%), rock powder (31.11%) and glucose (14.9%), as well as the quadratic effect of temperature (10.28%), were the strongest parameters, being responsible for a total of 90.3% of phosphate solubilization (Figure 2C).

Pareto analysis showed that, for pH, the largest effects were the quadratic effect of temperature (27.52%) and the linear effect of glucose (26.14%), followed by the S × R interaction (22.01%) and the linear effect of rock powder (10.75%) (Figure 3A).

Figure 3
Percentage effect of significant parameters estimated by Pareto analysis of the central composite design model for pH (a), acidity (b), and P solubilization (c), T – Temperature; S – glucose; R – rock powder.

For acidity, the linear effect of temperature showed the greatest contribution (19.82%), while the quadratic effects of temperature (7.80%) and glucose (4.92%) showed the smallest contributions; the other parameters (S, R, R², T × S and S × R) varied between 11.59% and 15.37% (Figure 3B).

For solubilized phosphorus, the linear effect of temperature (34.01%), rock powder (31.11%), and glucose (14.9%), as well as the quadratic effect of temperature (10.28%), were the strongest parameters, accounting for 90.3% of phosphate solubilization (Figure 3C).

To evaluate the accuracy of the models derived from the central composite design. after excluding insignificant terms (Equations 6, 7, and 8), the measured values ​​of the response variables - pH, acidity, and solubilized phosphorus - evaluated in each experiment (Table 2) were plotted against the quantities fitted by the models (Figure 3). The results show that the model appropriately estimated these variables, accounting for 94%, 92%, and 94% of the variation in pH (Figure 4A), acidity content (Figure 4B), and P solubilization (Figure 4C), respectively.

Figure 4
Curve of actual values ​​versus predicted pH (a), acidity (b), and soluble phosphate (c).

Three-dimensional (3D) surface plots were generated from the fitted polynomial equation and illustrate the most significant interactive effects of the independent variables on pH, acidity, and phosphate solubility. Figures 5 and 6 present the most relevant parameters of each response, keeping one variable constant at the optimal level while the others vary within their experimental ranges. For pH, the lowest values (4.36 and 4.47) were observed under the following conditions: 64.93 g L⁻¹ glucose, 30.37 °C, and 8.50 g L⁻¹ rock powder (Figure 5A), and 29.76 °C, 35.5 g L^-1 glucose, and 14.38 g L^-1 rock powder (Figure 5B). When rock powder and glucose concentrations were reduced to 2.61 and 6.07 g L^-1, respectively, the pH reached 5.98 (Figure 5C).

Figure 5
Response surface and contour plot for pH as a function of temperature and glucose variation with rock powder constant at the center point (a); temperature and rock concentration with glucose constant at the center point (b); glucose and rock concentration with constant temperature at the center point (c); and variation in acidity concentration as a function of temperature and glucose variation with rock powder constant at the center point (d); temperature and rock concentration with glucose constant at the center point (e); glucose and rock concentration with constant temperature at the center point (f).

Figure 6
Response surface and contour plot for phosphorus content as a function of temperature and glucose variation with rock powder at a constant center point (a), temperature and rock concentration with glucose at a constant center point (b), and glucose and rock concentration with constant temperature at a constant center point (c).

For acidity, a value of 139.57 mg L^-1 was obtained at 28.70 °C and 22.12 g L^-1 glucose with 8.5 g L^-1 rock powder (Figure 5D). A region of high acidity (196.13 mg L^-1) was observed at 28.70 °C and 2.61 g L^-1 rock powder with constant glucose (35.5 g L⁻¹) (Figure 5E). Acidity reached 193.76 mg L^-1 under 64.93 g L^-1 glucose and 2.61 g L^-1 rock powder (Figure 5F).

Regarding phosphate solubilization, the phosphorus concentration in the culture medium increased with the addition of glucose but was inversely proportional to temperature and rock powder concentration (Equation 8). No interaction between factors showed a significant effect, although all quadratic effects were relevant, with temperature being the most influential factor (p < 0.01) (Table 3).

For phosphate solubilization, maximum phosphorus concentrations of 40.92 mg L^-1 (Figure 6A), 42.45 mg L^-1 (Figure 6B), and 54.72 mg L^-1 (Figure 6C) were observed under different combinations of glucose, temperature, and rock powder. The optimal operational parameters predicted by the model for maximum phosphate solubilization (57.73 mg L^-1) were 28.35 °C, 64.93 g L^-1 glucose, and 2.61 g L^-1 rock phosphate. Under these conditions, pH and titratable acidity were 4.31 and 213.77 mg L^-1, respectively.

4. Discussion

The formation of a translucent halo around the colonies indicates the ability of the microorganism to release metabolites capable of solubilizing poorly soluble phosphate compounds present in the culture medium (Figure 1). This phenomenon is generally associated with the production of organic acids, which promote phosphorus solubilization through acidification and chelation processes. Proton release favors the protonation of phosphate groups and the displacement of cations bound to the mineral matrix, while the formation of complexes between organic acids and metallic ions, such as Ca²⁺, Fe³⁺, and Al³⁺, weakens metal phosphate bonds, contributing to the release of phosphorus in soluble forms (Salisu and Isiya, 2024; Amri et al., 2023).

In the present study, the phosphate solubilization index (PSI) was 2.72, which is lower than the value reported by Joshi et al. (2023), who observed a PSI of 4.75 ± 0.06 for isolate L4, accompanied by quantitative solubilization of 891.38 ± 18.55 μg mL⁻¹. This difference highlights the metabolic variability among strains of the genus Pseudomonas, which is often attributed to differences in metabolic pathways involved in organic acid production, as well as to the influence of environmental factors such as pH, temperature, soil type, and nutrient availability (Guardiola-Márquez et al., 2023; Joshi et al., 2023; Qingwei et al., 2023; Janati et al., 2023).

Although halo formation in solid media is widely used as a preliminary criterion for screening phosphate-solubilizing bacteria, this visual indicator does not necessarily reflect the full metabolic potential of the microorganism. Some bacteria may exhibit significant solubilization capacity without producing visible halos, reinforcing the need for complementary quantitative evaluations (Elhaissoufi et al., 2022). Therefore, the combination of qualitative observations in solid media and quantitative determination of solubilized phosphorus in liquid media provides a more robust approach for assessing solubilization activity.

The inverse relationship observed between pH reduction, increased acidity, and the increase in soluble phosphorus concentration indicates that the solubilization promoted by Pseudomonas salmasensis is strongly associated with medium acidification (Figures 2A, 2B, 2C). Species of Pseudomonas are known to produce various organic acids capable of mobilizing phosphorus from insoluble minerals, among which gluconic acid stands out due to its high calcium-complexing capacity. Studies with Pseudomonas aeruginosa have reported the production of acids such as gluconic, succinic, oxalic, and citric acids, which contribute both to pH reduction and to the complexation of cations associated with phosphate minerals (Ahmad et al., 2022a). Other metabolites, including malonic, lactobionic, and glutamic acids, have also been associated with the dissolution of mineral phosphates (Xu et al., 2025).

The regression coefficients obtained in the experimental model also provide insights into the role of the evaluated factors in the solubilization dynamics (Equations 6, 7, and 8). For pH, the negative linear effects of glucose and rock powder suggest that increased carbon availability stimulates bacterial metabolism and the production of acidic metabolites (Equation 6). Readily assimilable carbon sources provide energy and reducing equivalents required for metabolic pathways involved in organic acid biosynthesis. Metabolomic evidence indicates that Pseudomonas strains can produce compounds such as malonic and lactobionic acids during tricalcium phosphate solubilization (Xu et al., 2025).

The rock powder used in this study as a phosphate source also influenced pH dynamics, suggesting that minerals present in rock powders may exert a buffering effect, modulating the acidification promoted by microorganisms (Damor and Goswami, 2024; Zhang et al., 2024; Tariq and Ahmed, 2023). This effect becomes evident in the response observed for acidity, in which most coefficients exhibited negative effects, except for the quadratic term associated with rock powder (Equation 7). This behavior suggests that solubilization efficiency depends on the balance between microbial acid production and the chemical resistance of the mineral matrix (Amri et al., 2023; Tariq and Ahmed, 2023).

For soluble phosphorus, the negative linear effects of temperature and rock powder concentration indicate that high values of these factors may limit process efficiency (Equation 8). The solubilization activity of Pseudomonas strains is temperature-dependent, as different isolates present specific optimal ranges for growth and metabolic activity. In some cases, moderate temperatures favor the production of metabolites involved in solubilization, whereas higher values may reduce this activity (Dasila et al., 2023; Ahmad et al., 2022b).

The concentration of the mineral source also influences solubilization efficiency in a non-linear manner. At high concentrations of phosphate rock, the increased mineral load may reduce process efficiency, possibly due to system saturation or the presence of minerals with a neutralizing effect on medium acidification (Barin et al., 2022; Xie et al., 2021; Buss et al., 2023).

Optimization studies using response surface methodology have been widely applied to improve phosphate solubilization efficiency by microorganisms, including studies with Pseudomonas putida Rs-198 and other rhizobacteria (Peng et al., 2014; Lebrazi et al., 2020). Although the soluble phosphorus values observed in this study were lower than those reported by Waday et al. (2022), they were comparable to those obtained by Peng et al. (2014). These differences among studies may be related to the physiological characteristics of the isolates, experimental conditions, and the chemical composition of the phosphate sources used.

5. Conclusion

Phosphate solubilization by Pseudomonas salmasensis CFB 004 was significantly influenced by the interaction between temperature, glucose concentration, and rock powder.

The best operating conditions for phosphate solubilization (57.73 mg L^-1) were: a temperature of 28.35 °C, glucose at 64.93 g L^-1, and rock powder at 2.61 g L^-1, in which the medium had a pH of 4.31 and a titratable acidity of 213.77 mg L^-1.

Glucose was crucial for acidifying the medium and, consequently, for increasing phosphorus solubilization, while higher concentrations of rock powder exerted a buffering effect limiting the reduction in pH and the release of the nutrient.

Acknowledgements

The authors acknowledge the Postgraduate Program in Agricultural Engineering, the Semi-Arid Agrobiodiversity Research Group and the Institutional Scientific Initiation Scholarship Program PIBIC/UEPB/CNPq, Grant No. 03/2023. PROGRAMA INSTITUCIONAL DE BOLSAS DE INICIAÇÃO CIENTÍFICA UEPB/CNPq

Data Availability Statement

All the data supporting the results of this study were published in the article itself.

References

• AHMAD, A., MOIN, S.F., LIAQAT, I., SALEEM, S., MUHAMMAD, F., MUJAHID, T. and ZAFAR, U., 2022a. Isolation, solubilization of inorganic phosphate, and production of organic acids by individual and co-inoculated microorganisms. Geomicrobiology Journal, vol. 40, no. 1, pp. 111-121. https://doi.org/10.1080/01490451.2022.2124329
  » https://doi.org/10.1080/01490451.2022.2124329
• AHMAD, A., ZAFAR, U., KHAN, A., HAQ, T., MUJAHID, T. and WALI, M., 2022b. Effectiveness of compost inoculated with phosphate solubilizing bacteria. Journal of Applied Microbiology, vol. 133, no. 2, pp. 1115-1129. https://doi.org/10.1111/jam.15633 PMid:35603602.
  » https://doi.org/10.1111/jam.15633
• AL-TAMMAR, F.K. and KHALIFA, A.Y.Z., 2022. Plant growth promoting bacteria drive food security. Brazilian Journal of Biology, vol. 82, pp. e267257. https://doi.org/10.1590/1519-6984.267257 PMid:36515299.
  » https://doi.org/10.1590/1519-6984.267257
• AMERICAN PUBLIC HEALTH ASSOCIATION - APHA. AWWA. WEF, 1998a. Standard Methods for the Examination of Water and Wastewater 20th ed. Washington, D.C.: American Public Health Association. Part 2000: Physical & Aggregate Properties. 2310 Acidity.
• AMERICAN PUBLIC HEALTH ASSOCIATION – APHA. AWWA. WEF, 1998b. Standard Methods for the Examination of Water and Wastewater 20th ed. Washington, D.C.: American Public Health Association. Part 4000: Inorganic Nonmetallic Constituents. 4500-H pH value.
• AMRI, M., MATEUS, D., GATROUNI, M., RJEIBI, M.R., ASSES, N. and ABBES, C., 2022. Co-inoculation with phosphate-solubilizing microorganisms of rock phosphate and phosphogypsum and their effect on growth promotion and nutrient uptake by ryegrass. Applied Biosciences, vol. 1, no. 2, pp. 179-197. https://doi.org/10.3390/applbiosci1020012
  » https://doi.org/10.3390/applbiosci1020012
• AMRI, M., RJEIBI, M.R., GATROUNI, M., MATEUS, D.M.R., ASSES, N., PINHO, H.J.O. and ABBES, C., 2023. Isolation, identification, and characterization of phosphate-solubilizing bacteria from Tunisian soils. Microorganisms, vol. 11, no. 3, pp. 783. https://doi.org/10.3390/microorganisms11030783 PMid:36985356.
  » https://doi.org/10.3390/microorganisms11030783
• BARIN, M., ASADZADEH, F., HOSSEINI, M., HAMMER, E.C., VETUKURI, R.R. and VAHEDI, R., 2022. Optimization of biofertilizer formulation for phosphorus solubilizing by Pseudomonas fluorescens Ur21 via response surface methodology. Processes (Basel, Switzerland), vol. 10, no. 4, pp. 650. https://doi.org/10.3390/pr10040650
  » https://doi.org/10.3390/pr10040650
• BUSS, W., HASEMER, H., FERGUSON, S. and BOREVITZ, J., 2023. Stabilisation of soil organic matter with rock powder partially counteracted by plants. Global Change Biology, vol. 30, no. 1, pp. e17052. https://doi.org/10.1111/gcb.17052 PMid:37994295.
  » https://doi.org/10.1111/gcb.17052
• CHAGAS JÚNIOR, A.F., OLIVEIRA, L.A., OLIVEIRA, A.N. and WILLERDING, A.L., 2010. Capacidade de solubilização de fosfatos e eficiência simbiótica de rizóbios isolados de solos da Amazônia. Acta Scientiarum. Agronomy, vol. 32, no. 2, pp. 359-366. https://doi.org/10.4025/actasciagron.v32i2.3185
  » https://doi.org/10.4025/actasciagron.v32i2.3185
• DAMOR, S. and GOSWAMI, P., 2024. Bacterial strain improvement via random physical mutation to improve phosphate solubilization efficiency for sustainable crop growth. Journal of Scientific Research, vol. 16, no. 1, pp. 243-251. https://doi.org/10.3329/jsr.v16i1.64892
  » https://doi.org/10.3329/jsr.v16i1.64892
• DASILA, H., SAH, V.K., JAGGI, V., KUMAR, A., TEWARI, L., TAJ, G., CHATURVEDI, S., PERVEEN, K., BUKHARI, N.A., SIANG, T.C. and SAHGAL, M., 2023. Cold-tolerant phosphate-solubilizing Pseudomonas strains promote wheat growth and yield by improving soil phosphorous (P) nutrition status. Frontiers in Microbiology, vol. 14, pp. 1135693. https://doi.org/10.3389/fmicb.2023.1135693 PMid:37025630.
  » https://doi.org/10.3389/fmicb.2023.1135693
• DIAS, R.D.C., STAFANATO, J.B., ZONTA, E., POLIDORO, J.C., GONÇALVES, R.G.D.M. and TEIXEIRA, P.C., 2022. Effect of phosphate organomineral fertilization on the dry matter production and phosphorus accumulation of corn. Journal of Agricultural Science (Toronto), vol. 14, no. 8, pp. 78-87. https://doi.org/10.5539/jas.v14n8p78
  » https://doi.org/10.5539/jas.v14n8p78
• ELHAISSOUFI, W., IBNYASSER, A., HADDINE, M., ZEROUAL, Y., GHANI, R., BARAKAT, A., RCHIAD, Z., GHOULAM, C. and BARGAZ, A., 2022. Screening of potential phosphate solubilizing bacteria inoculants should consider the contrast in phosphorus bio-solubilization rate along with plant growth promotion and phosphorus use efficiency. Journal of Applied Microbiology, vol. 134, no. 2, pp. lxac077. https://doi.org/10.1093/jambio/lxac077 PMid:36724266.
  » https://doi.org/10.1093/jambio/lxac077
• GHORESHIZADEH, S., CALVO-PEÑA, C., RUIZ-MUÑOZ, M., OTERO-SUÁREZ, R., COQUE, J.J.R. and COBOS, R., 2024. Pseudomonas taetrolens ULE-PH5 and Pseudomonas sp. ULE-PH6 isolated from the hop rhizosphere increase phosphate assimilation by the plant. Plants, vol. 13, no. 3, pp. e402. https://doi.org/10.3390/plants13030402 PMid:38337935.
  » https://doi.org/10.3390/plants13030402
• GUARDIOLA-MÁRQUEZ, C.E., SANTOS-RAMÍREZ, M.T., FIGUEROA-MONTES, M.L., VALENCIA-DE LOS COBOS, E.O., STAMATIS-FÉLIX, I.J., NAVARRO-LÓPEZ, D.E. and JACOBO-VELÁZQUEZ, D.A., 2023. Identification and characterization of beneficial soil microbial strains for the formulation of biofertilizers based on native plant growth-promoting microorganisms isolated from northern Mexico. Plants, vol. 12, no. 18, pp. e3262. https://doi.org/10.3390/plants12183262 PMid:37765426.
  » https://doi.org/10.3390/plants12183262
• JANATI, W., BOUABID, R., MIKOU, K., GHADRAOUI, L.E. and ERRACHIDI, F., 2023. Phosphate solubilizing bacteria from soils with varying environmental conditions: occurrence and function. PLoS One, vol. 18, no. 12, pp. e0289127. https://doi.org/10.1371/journal.pone.0289127 PMid:38064520.
  » https://doi.org/10.1371/journal.pone.0289127
• JOHAN, P.D., AHMED, O.H., OMAR, L. and HASBULLAH, N.A., 2021. Phosphorus transformation in soils following co-application of charcoal and wood ash. Agronomy (Basel), vol. 11, no. 10, pp. e2010. https://doi.org/10.3390/agronomy11102010
  » https://doi.org/10.3390/agronomy11102010
• JOSHI, S., GANGOLA, S., JAGGI, V. and SAHGAL, M., 2023. Functional characterization and molecular fingerprinting of potential phosphate solubilizing bacterial candidates from Shisham rhizosphere. Scientific Reports, vol. 13, no. 1, pp. 7003. https://doi.org/10.1038/s41598-023-33217-9 PMid:37117212.
  » https://doi.org/10.1038/s41598-023-33217-9
• KHAN, K.S., NAVEED, M., QADIR, M.F., YASEEN, M. and SIDDIQUI, M.H., 2023. Bio-organically acidified product-mediated improvements in phosphorus fertilizer utilization, uptake and yielding of Zea mays in calcareous soil. Plants, vol. 12, no. 17, pp. e3072. https://doi.org/10.3390/plants12173072 PMid:37687319.
  » https://doi.org/10.3390/plants12173072
• LEBRAZI, S., NIEHAUS, K., BEDNARZ, H., FADIL, M., CHRAIBI, M. and FIKRI-BENBRAHIM, K., 2020. Screening and optimization of indole-3-acetic acid production and phosphate solubilization by rhizobacterial strains isolated from Acacia cyanophylla root nodules and their effects on its plant growth. Journal of Genetic Engineering and Biotechnology, vol. 18, no. 1, pp. 71. https://doi.org/10.1186/s43141-020-00090-2 PMid:33175273.
  » https://doi.org/10.1186/s43141-020-00090-2
• LUO, D., SHI, J., LI, M., CHEN, J., WANG, T., ZHANG, Q., YANG, L., ZHU, N. and WANG, Y., 2024. Consortium of phosphorus-solubilizing bacteria promotes maize growth and changes the microbial community composition of rhizosphere soil. Agronomy (Basel), vol. 14, no. 7, pp. e1535. https://doi.org/10.3390/agronomy14071535
  » https://doi.org/10.3390/agronomy14071535
• MARCOTTE, A.R., ANBAR, A.D., MAJESTIC, B.J. and HERCKES, P., 2020. Mineral powder and iron solubility: effects of composition, particle size, and surface area. Atmosphere (Basel), vol. 11, no. 5, pp. 533. https://doi.org/10.3390/atmos11050533
  » https://doi.org/10.3390/atmos11050533
• NAUTIYAL, C.S., 1999. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters, vol. 170, no. 1, pp. 265-270. https://doi.org/10.1111/j.1574-6968.1999.tb13383.x PMid:9919677.
  » https://doi.org/10.1111/j.1574-6968.1999.tb13383.x
• OLIVEIRA, C.L.B.D., CASSIMIRO, J.B., LIRA, M.V.D.S., BONI, A.S., DONATO, N.L., REIS JUNIOR, R.A. and HEINRICHS, R., 2022. Sugarcane ratoon yield and soil phosphorus availability in response to enhanced efficiency phosphate fertilizer. Agronomy (Basel), vol. 12, no. 11, pp. 2817. https://doi.org/10.3390/agronomy12112817
  » https://doi.org/10.3390/agronomy12112817
• PENG, Y., HE, Y., WU, Z., LU, J. and LI, C., 2014. Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM. Brazilian Journal of Microbiology : Publication of the Brazilian Society for Microbiology, vol. 45, no. 4, pp. 1229-1237. https://doi.org/10.1590/S1517-83822014000400013 PMid:25763026.
  » https://doi.org/10.1590/S1517-83822014000400013
• QINGWEI, Z., LUSHI, T., YU, Z., YU, S., WANTING, W., JIANGCHUAN, W., XIAOLEI, D., XUEJIAO, H. and BILAL, M., 2023. Isolation and characterization of phosphate-solubilizing bacteria from rhizosphere of poplar on road verge and their antagonistic potential against various phytopathogens. BMC Microbiology, vol. 23, no. 1, pp. 221. https://doi.org/10.1186/s12866-023-02953-3 PMid:37580699.
  » https://doi.org/10.1186/s12866-023-02953-3
• SALISU, B. and ISIYA, S., 2024. Biofertilizer production using phosphate-solubilizing Pseudomonas spp. isolated from rhizosphere soil: towards indigenous biofertilizer for enhanced crop productivity in Katsina, Nigeria. UMYU Journal of Microbiology Research (Rosemead, Calif.), vol. 9, no. 1, pp. 123-133. https://doi.org/10.47430/ujmr.2491.014
  » https://doi.org/10.47430/ujmr.2491.014
• SANCHEZ-GONZALEZ, M.E., MORA-HERRERA, M.E., WONG-VILLARREAL, A., DE LA PORTILLA-LÓPEZ, N., SANCHEZ-PAZ, L., LUGO, J., VACA-PAULÍN, R., DEL AGUILA, P. and YAÑEZ-OCAMPO, G., 2023. Effect of pH and carbon source on phosphate solubilization by bacterial strains in Pikovskaya medium. Microorganisms, vol. 11, no. 1, pp. e49. https://doi.org/10.3390/microorganisms11010049 PMid:36677341.
  » https://doi.org/10.3390/microorganisms11010049
• SHARMA, P., PANDEY, R. and CHAUHAN, N.S., 2024. Unveiling wheat growth promotion potential of phosphate solubilizing Pantoea agglomerans PS1 and PS2 through genomic, physiological, and metagenomic characterizations. Frontiers in Microbiology, vol. 15, pp. e1467082. https://doi.org/10.3389/fmicb.2024.1467082 PMid:39318437.
  » https://doi.org/10.3389/fmicb.2024.1467082
• TARIQ, A. and AHMED, A. 2023. Phosphate solubilization potential of PSB: an advance approach to enhance phosphorous availability for phytostimulation. https://doi.org/10.21203/rs.3.rs-3463966/v1
  » https://doi.org/10.21203/rs.3.rs-3463966/v1
• TEIXEIRA, P.C., DONAGEMMA, G.K., FONTANA, A. and TEIXEIRA, W.G., 2017. Manual de métodos de análise de solo 3. ed. Brasília: Embrapa, 573 p.
• VEY, R.T., JACQUES, R.J.S., PAVINATO, P.S., LÓPEZ-BAENA, F.J., SPANEVELLO, J.F., BRONZATTO, E.S., FULANETI, F.S., SANTOS, R.E., BRUM, L.N. and MARTIN, T.N., 2025. Phosphate solubilization capacity by bacteria in soybean crops. Brazilian Journal of Biology, vol. 85, pp. e289630. https://doi.org/10.1590/1519-6984.289630 PMid:40638407.
  » https://doi.org/10.1590/1519-6984.289630
• WADAY, Y.A., AKLILU, E.G., BULTUM, M.S. and ANCHA, V.R., 2022. Optimization of soluble phosphate and IAA production using response surface methodology and ANN approach. Heliyon, vol. 8, no. 12, pp. e12224. https://doi.org/10.1016/j.heliyon.2022.e12224 PMid:36582684.
  » https://doi.org/10.1016/j.heliyon.2022.e12224
• XIE, J., YAN, Z., WANG, G., XUE, W., LI, C., CHEN, X. and CHEN, D., 2021. A bacterium isolated from soil in a karst rocky desertification region has efficient phosphate-solubilizing and plant growth-promoting ability. Frontiers in Microbiology, vol. 11, pp. 625450. https://doi.org/10.3389/fmicb.2020.625450 PMid:33597933.
  » https://doi.org/10.3389/fmicb.2020.625450
• XU, Q., ZHAO, Y., HU, C., WU, S., TAN, Q. and SUN, X. 2025. Integrated mineralogical, metabolomic, and gene expression analysis of the phosphate-solubilizing mechanism of Pseudomonas sp. PSB-13. https://doi.org/10.21203/rs.3.rs-8305279/v1
  » https://doi.org/10.21203/rs.3.rs-8305279/v1
• ZHANG, Y., XU, T., FU, H. and WANG, Y., 2024. The impact of phosphate solubilizing bacteria modified biochar on maize seedling growth and rhizosphere phosphate availability. Polish Journal of Environmental Studies, vol. 33, no. 3, pp. 3463-3470. https://doi.org/10.15244/pjoes/186000
  » https://doi.org/10.15244/pjoes/186000
• ZHOU, L., SU, L., ZHANG, L., ZHANG, L., ZHENG, Y. and TANG, L., 2022. Effect of different types of phosphate fertilizer on phosphorus absorption and desorption in acidic red soil of Southwest China. Sustainability (Basel), vol. 14, no. 16, pp. 9973. https://doi.org/10.3390/su14169973
  » https://doi.org/10.3390/su14169973

*

e-mail: [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  14 Sept 2026
• Date of issue
  2026

History

• Received
  06 Apr 2026
• Accepted
  14 Aug 2026