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Article

Bacillus safensis P1.5S Exhibits Phosphorus-Solubilizing Activity Under Abiotic Stress

by
Loredana-Elena Mantea
,
Amada El-Sabeh
,
Marius Mihasan
and
Marius Stefan
*
Department of Biology, Faculty of Biology, Alexandru Ioan Cuza University of Iasi, Bd. Carol I, No. 11, 700506 Iasi, Romania
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 388; https://doi.org/10.3390/horticulturae11040388
Submission received: 26 February 2025 / Revised: 31 March 2025 / Accepted: 2 April 2025 / Published: 5 April 2025
(This article belongs to the Special Issue Plant–Microbial Interactions: Mechanisms and Impacts)
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Abstract

:
Climate change significantly impacts plant growth by reducing the availability of essential nutrients, including phosphorus (P). As an alternative to chemical fertilizers, climate-smart agriculture should prioritize the use of beneficial microorganisms such as P-solubilizing bacteria (PSB). Here, we report the ability of the P1.5S strain of Bacillus safensis to solubilize P under the stress caused by different pH, temperature, and salinity. Genomic data and the TBLASTN algorithm were used to identify genes involved in stress tolerance and P solubilization. Stress tolerance was confirmed by cultivation under varying conditions, while the mechanism of P solubilization was investigated using HPLC. Bioinformatic analysis revealed at least 99 genes related to stress tolerance, 32 genes responsible for organic acids synthesis, as well as 10 genes involved in phosphatase production. B. safensis P1.5S can grow at 37 °C, high NaCl concentrations (15 g/L), and is tolerant of alkaline and acidic conditions. The P1.5S strain primarily solubilizes P by releasing organic acids, including lactic, acetic, and succinic acid. Our data revealed that the efficacy of P solubilization was not affected by abiotic stressors (19.54 µg P/mL). By evaluating the P solubilization ability of B. safensis P1.5S induced by stressors represented by varying pH, temperature, and salinity conditions, this work introduces a new avenue for increasing P availability, which enables and endorses the future development of practical applications of B. safensis P1.5S in challenging agricultural environments.

1. Introduction

Food production, one of the greatest challenges facing the agricultural sector, is influenced by a wide variety of factors such as climate and weather conditions, and soil quality and fertility [1]. Changing weather patterns or extreme weather events can damage crops or disrupt harvests [2], threatening global food security [3]. Unpredictable climate changes are associated with different abiotic stresses such as alkalinity, acidity, salinity, drought, low and high temperatures, representing a major risk for sustainable agriculture [4]. Climate changes affect not only food quality and crop yields, but also the microbial population and their enzymatic activities in soil, reducing the availability and uptake of important nutrients for plant growth [3,5,6].
Phosphorus (P) is one of the most important macronutrients, essential for plant growth and development [7]. However, due to its poor solubility, P availability can be limited in many soils, with significant effects on plant growth and agricultural productivity. To support crop growth and sustain high yields, additional P is supplied to agricultural soils in the form of mineral or organic fertilizers [8]. However, because of the fixation of P as iron or aluminum phosphates in acid soils, or as calcium phosphate in neutral and alkaline soils, only a small portion of the applied P is available for plant uptake. Moreover, P is considered a non-renewable resource and, due to the high rate of use, it is estimated that phosphate rock reserves used in fertilizer production could be significantly depleted within the next 50 to 100 years [9]. As the widespread use of P fertilizers is expensive, unsustainable, and a source of pollution, an environmentally friendly approach is needed in the current context of climate change [10].
An alternative approach to chemical fertilizer is the use of phosphorus-solubilizing microorganisms (PSMs). Naturally, PSMs play an important role in soil P dynamics and availability, converting insoluble phosphorus compounds into soluble forms accessible to plants using various mechanisms. One of the most important mechanisms involves the production of low molecular weight organic acids and extracellular enzymes [11]. PSMs are an emerging biotechnological tool in agriculture and environmental engineering, due to their advantages such as eco-friendliness, low production cost, and high efficiency [10,12]. Phosphate solubilizing bacteria (PSB) inoculated on seeds or soil improved solubilization of bound soil phosphorus and applied phosphates, resulting in higher plant performance [12,13,14]. Although several studies have demonstrated the potential of PSB to enhance plant growth and improve soil fertility, their effectiveness in the field is highly influenced by climate, soil type and other environmental factors [13]. Different stressors such as alkalinity, acidity, salinity and extreme temperatures cause changes in the abundance, diversity and functionality of PSB populations [15]. Therefore, bacteria with phosphorus-solubilizing potential must be tolerant to abiotic stress to be suitable for agricultural applications.
The genus Bacillus is among the most important soil bacterial groups, playing a key role in improving soil fertility and promoting plant growth, particularly through phosphorus solubilization [16]. Bacillus species (including B. subtilis, B. megaterium, B. safensis and others) are widely recognized for their ability to enhance plant growth under various stressful environmental conditions, making them important in the context of stress tolerance [17]. Among Bacillus species, B. safensis has gained attention for its potential to enhance the growth of various plants (e.g., potato, tomato, and radish) and improve their tolerance to abiotic stress [18,19,20,21]. Although its tolerance to drought, salinity, heat stress, and heavy metal toxicity is well-documented [22,23,24], to the best of the authors’ knowledge, its ability to solubilize mineral phosphates, particularly under abiotic stress conditions, remains largely unexplored. In this context, the objective of our research was to evaluate the ability of a Bacillus safensis strain isolated from phosphorus-deficient soils to solubilize inorganic P under different stress conditions related to pH, temperature and salinity. Genomic data were initially employed to evaluate the strain’s capacity for mineral phosphate solubilization and its tolerance to abiotic stress. Furthermore, the production of organic acids and both alkaline and acid phosphatases was assessed to better understand the mechanisms of P solubilization. Our findings demonstrate that Bacillus safensis P1.5S effectively solubilizes phosphorus under stress conditions, highlighting its potential as a plant growth-promoting bacterium. However, further field trials are necessary to assess the suitability of strain P1.5S for practical applications in challenging agricultural environments.

2. Materials and Methods

2.1. Bacterial Strain and Culture Media

The strain designated as P1.5S was isolated by our research team in 2018 from phosphorus-deficient soil located in the north-eastern part of Iasi County, Romania, and deposited in the culture collection of the Microbiology Laboratory, Alexandru Ioan Cuza University of Iasi under the name Bacillus safensis P1.5S. Pikovskaya (PVK) agar (glucose, 7.5 g; (NH4)2SO4, 0.375 g; NaCl, 0.150 g; MgSO4 7H2O, 0.075 g; MnSO4, 0.0015 g; KCl, 0.150 g; yeast extract, 0.375 g; Ca3(PO4)2, 2.250 g; agar, 18 g; H2O, 1 L) containing tri-calcium phosphate (TCP) as an insoluble phosphorus source was used as the selective medium [25]. Following isolation, the strain was stored at −80 °C in 30% glycerol stocks. Luria Bertani (LB) agar (Roth, Karlsruhe, Germany) was used for cultivation at 28 °C for 48 h. One colony considered typical for the P1.5S strain was inoculated in 10 mL LB broth (Roth, Karlsruhe, Germany) and cultivated overnight at 28 °C in aerobic conditions, to serve as an inoculum for further experiments. PVK broth was used for the assessment of P solubilization potential and LB broth for the evaluation of bacterial growth under stress conditions.

2.2. Bioinformatic Analysis of the Draft Genome of Strain P1.5S

The draft genome of the P1.5S strain (NCBI BioProject PRJNA960951, Assembly GCA_029930515.1, WGS JARZFW000000000) was assessed with CheckM v1.2.2 for completeness and contamination [26], uploaded to the RAST server (Rapid Annotation using Subsystem Technology) for functional annotation [27] and to the Type (Strain) Genome Server (TYGS) for a whole genome-based taxonomic analysis [28]. The TBLASTN algorithm was used to identify genes involved in P solubilization (organic acids and phosphatases production) and genes conferring tolerance to abiotic stress [29]. Genes with an identity higher than 30% and a query coverage value above 70% were considered positive hits. A database of UniProt protein sequences of interest was created and subsequently searched against the draft genome [30]. For all tools and servers, default parameters were used unless otherwise stated.

2.3. Evaluation of Bacterial Growth Under Abiotic Stress Conditions

The growth of the P1.5S strain was evaluated under different stress conditions related to alkalinity, acidity, salinity and temperature. The optical density (OD600) of the inoculum, obtained as outlined above, was adjusted to approximately 0.100 using a DU 730 spectrophotometer (Beckman Coulter, Washington D.C., USA). To serve as control, 1 mL of the inoculum was transferred to 25 mL of LB medium (pH 7) containing 10 g of NaCl, and incubated at 28 °C, 190 rpm, for 72 h. Acidic and alkaline stress was simulated by adjusting the culture medium pH to 5 and 9, respectively. For salt stress conditions, the strain was cultivated in LB broth containing either 2.5 g/L or 15 g/L NaCl. To assess the effect of temperature, P1.5S cultures were incubated at 20 or 37 °C.
To evaluate the strain’s growth under various conditions, samples were taken every hour until 12 h, and then at 24, 48 and 72 h and the optical densities (OD at 600 nm) were determined spectrophotometrically. A growth curve was constructed by plotting the mean optical densities over time.

2.4. Phosphate Solubilization Under Abiotic Stress Conditions

Quantification of soluble P released from TCP was performed following the procedure outlined by [31], with minor modifications. To prepare the inoculum, an overnight culture in LB broth was centrifuged at 4500 rpm for 15 min. The supernatant was discarded, the bacterial cells resuspended in sterile distilled water and the OD600 adjusted to 0.100. One mL of this suspension was used to inoculate flasks containing 25 mL PVK broth. Uninoculated PVK served as negative control for soluble P quantification.
The ability of the P1.5S strain to solubilize TCP was evaluated under temperature, pH and salinity stress. For alkaline conditions, the pH of the PVK medium was adjusted to 9. The effect of salinity was investigated using PVK broth containing different NaCl concentrations: 2.5 g/L, 10 g/L and 15 g/L. To assess the influence of temperature, bacterial cultures were incubated at 20 °C and 37 °C. The experimental control was represented by P1.5S cultivated at 28 °C in PVK medium (pH 7), containing 0.150 g/L NaCl. Negative controls (uninoculated PVK) were prepared for all tested stress conditions. All samples and controls were incubated for 10 days at 28 °C and 190 rpm, except those used for the evaluation of temperature effects.
Samples were aseptically removed at 3, 5, 7 and 10 days after inoculation (DAI) and used for the determination of soluble P, organic acids production and acid and alkaline phosphatase activity. Briefly, 1 mL from each culture was centrifuged at 14,000 rpm for 30 min and the quantity of soluble P was determined by a colorimetric procedure using the molybdenum blue reaction [32]. The pH of each sample was measured using a Hanna HI 2211 pH meter (Hanna Instruments, Rhode Island, USA). To evaluate the growth of the P1.5S strain in PVK medium under different P solubilization conditions, each sample was subjected to a colony forming units (CFU) count. Briefly, 0.1 mL samples were plated on DEV nutrient agar (Merck, Darmstadt, Germany) after performing a tenfold serial dilution. The plates were incubated for 48 h at 28 °C and the mean CFU/mL was calculated.

2.5. Determination of Organic Acids Production

Samples taken at 3, 5, 7 and 10 DAI were centrifuged at 14,000 rpm for 30 min and the supernatants were used for the identification of organic acids produced by P1.5S. Thus, 20 µL of each supernatant was injected into a PRP-X300 PSDVB column (Hamilton, Reno, NV, USA). Detection and quantification of organic acids was performed using a Bischoff HPLC system with a Lambda 1010 UV detector (Bischoff, Leonberg, Germany) and wavelength set at 210 nm. The eluent (mobile phase) used was 1 mN sulfuric acid at a flow rate of 1 mL/min. Sample retention time and peak area were compared with those of standards to identify the organic acids [33]. Each experiment was conducted in duplicate.

2.6. Acid and Alkaline Phosphatase Activity Assessment

Acid and alkaline phosphatase activities were evaluated using the method previously described by [34], with some modifications. Briefly, 300 µL of the supernatant obtained by centrifugation of the samples taken at 3, 5, 7 and 10 DAI was incubated for 3 h at 28 °C with 100 µL of 25 mM p-nitrophenol phosphate and 200 µL of modified universal buffer pH 6.5 and pH 11. After adding 100 µL of 0.5 M CaCl2 and 400 µL of 0.5 M NaOH, the mixture was centrifuged at 14,000 rpm for 10 min and the yellow color was measured at 410 nm. A calibration curve was constructed using p-nitrophenol (pNP) as the standard and the results were expressed as nmoles pNP/mL/h.

2.7. Statistical Analysis

Each experiment was repeated at least two times. The results were evaluated using Tukey’s and Sidak’s multiple comparisons tests. Also, the Pearson correlation coefficient was used to explore the relationships between solubilized P, CFU number, phosphatase activity and pH values. The data were analyzed using GraphPad Prism 9.3 software (GraphPad Software, Inc., La Jolla, CA, USA) and presented as mean ± SEM. Differences between groups were considered significant when p < 0.05.

3. Results

3.1. Functional Genome Analysis of Bacillus safensis P1.5S

According to the TYGS analyses, the P1.5S strain belongs to the Bacillus safensis species, with a digital DNA-DNA hybridization (dDDH) value of 80.6% when compared to B. safensis subsp. osmophilus CECT 9344T. The GCA_029930515.1 draft genome assembly of B. safensis P1.5S (99.22% estimated completeness and 0.29% estimated contamination by CheckM) contains 3749 genes, of which 3671 are protein-coding sequences [35]. The draft genome size is 3.6 Mb, falling within the 3.6 to 4.1 Mb genome size range described for the 35 complete Bacillus safensis genomes found in GenBank [36]. Figure 1 shows the subsystem statistics for B. safensis P1.5S. RAST analysis assigned a theoretical function to only 30% of the genes in the genome. The pie chart, generated and visualized with SEED Viewer [37], depicts the distribution of the most frequent subsystem categories among the 320 identified in the P1.5S genome. The most abundant subsystem categories were involved in the metabolism and transport of amino acids and derivatives (318 genes), carbohydrates (241 genes), and proteins (163 genes), as well as in the production of cofactors, vitamins, prosthetic groups, and pigments (154 genes). Additionally, genes involved in sporulation and dormancy (95 genes), nucleosides and nucleotides (91 genes), cell wall (70 genes), DNA (61 genes), RNA (55 genes) metabolism, and stress response (45 genes) were also identified (Figure 1).
Following BLAST analysis [38], several genes involved in phosphorus solubilization were identified in the genome of B. safensis P1.5S (Table S1). This strain presents at least 32 genes involved in the biosynthesis of organic acids, such as gluconic, formic, malic, citric, lactic, acetic, and succinic acids. In addition, 10 genes encoding the synthesis of acid and alkaline phosphatases were found with coverage ranging from 92% to 100%. Furthermore, BLAST analysis revealed the presence of at least 53 genes conferring tolerance to pH, temperature variation, salinity, and drought (Table S2). Moreover, in the B. safensis P1.5S genome were found 19 genes involved in the synthesis and transport of osmolytes, such as proline, glycine betaine, trehalose, and glutamate (Table S3). Also, the genome contains 27 genes that alleviate oxidative stress, including genes involved in bacillithiol and antioxidant enzymes synthesis (Table S4).

3.2. Effect of Temperature, pH and Salinity on Bacillus safensis P1.5S Growth

The ability of B. safensis P1.5S to grow in LB medium varies depending on the stress conditions. Regarding temperature, a growth delay of up to 6 h was observed during the incubation at 20 °C, compared with control (28 °C), for which a 4 h lag phase was recorded—Figure 2a. The maximum growth of the P1.5S strain cultured at 20 °C was reached after 24 h of incubation (OD600 = 0.964), while the highest optical density of control cultures was recorded after only 12 h (OD600 = 1.086). The incubation at 37 °C stimulated bacterial growth, inducing a shorter lag phase (2 h) and a maximum growth recorded after 12 h of incubation with a significantly higher optical density (OD600 = 1.501, p < 0.0001) compared with the control (Figure 2a). We must emphasize that incubation at 37 °C resulted in higher growth of the P1.5S strain, with significantly increased OD600 values recorded throughout the entire cultivation period compared with control.
Acidity (pH 5) and alkalinity (pH 9) negatively influenced the growth of B. safensis P1.5S. Thus, the results showed that the lag phase of the bacterial cells cultured in LB medium with pH adjusted to 5 or 9 was prolonged (up to 8 h), compared with the pH 7 control—Figure 2b. The maximum growth of cells cultured at pH 5 or 9 was observed at 24 h, whereas in the control cultures, it was reached after only 12 h of incubation. Significantly lower OD600 values were observed in alkaline growth conditions (p < 0.0001) (maximum value of 0.695) compared with neutral pH conditions (1.086). Our data revealed that between 24 and 72 h of incubation, the culture medium initially adjusted to pH 5 supported the growth of the P1.5S strain better than the alkaline conditions—Figure 2b.
No significant influence of salinity (2.5, 10, 15 g/L NaCl) on the growth of B. safensis P1.5S was observed during the entire incubation period, as shown in Figure 2c. However, one exception occurred at 24 h after inoculation when the maximum growth was recorded for the cells cultivated in LB supplemented with 2.5 g/L NaCl.

3.3. Phosphorus Solubilization Potential of Bacillus safensis P1.5S

Our results showed that B. safensis P1.5S had the ability to solubilize the insoluble phosphorus source from the PVK broth, namely Ca3(PO4)2. Over a 10-day period, the concentrations of soluble phosphorus ranged from 15.33 to 16.16 µg/mL, with no significant differences (p ≥ 0.05) between samples taken at 3, 5, 7, and 10 DAI—Figure 3. P solubilization was accompanied by a decrease of the initial culture medium pH (7.08), with the lowest value of 5.03 being measured at 3 DAI.
Pearson correlation analysis revealed a very strong negative relationship between solubilized P and pH values (r = −0.8449; p = 0.0716). A gradual increase of the viable cell number from 6.36 log10 CFU/mL at the beginning of the experiment to 8.38 log10 CFU/mL at 10 DAI was observed in the PVK medium. Data analysis indicated a very strong direct relationship (r = 0.8872, p = 0.0447) between the amount of solubilized phosphorus and the number of viable cells in the culture medium—Figure 3.

3.4. Bacillus safensis P1.5S Released Organic Acids and Phosphatases During P Solubilization

The production of organic acids was monitored at 3, 5, 7 and 10 days of incubation at 28 °C, following solubilized P quantifications and pH measurements. HPLC analysis revealed that B. safensis P1.5S produced eight organic acids: oxalic, citric, lactic, acetic, succinic, tartaric/gluconic, malic/formic and an unknown acid. Due to the retention time similarities, it was difficult to differentiate between tartaric/gluconic and malic/formic acids. The organic acids were released in the culture medium at different rates, depending on the incubation time—Figure 4a. For example, the concentration of succinic acid increased gradually after inoculation, from 1.97 mM at 3 DAI to 9.18 mM at 10 DAI. Acetic acid reached the maximum concentration (11.56 mM) after 10 DAI, but it was not evidenced at 5 DAI. Lactic acid concentration increased up to 5 DAI when the maximum value was recorded (8.03 mM) but significantly decreased at 7 DAI (p < 0.0001). The presence of citric acid was not evidenced in samples taken at 3 and 5 DAI, being detected in low concentrations only at 7 and 10 days (0.492 mM and 0.829 mM, respectively). Some organic acids such as succinic, acetic and lactic acids were produced at significantly higher concentrations compared with others (e.g., oxalic, tartaric/gluconic, malic/formic, and citric acids)—Figure 4a.
The activity of both the acid and alkaline phosphatase gradually increased up to 7 DAI, from 6.09 to 107.07 nmol pNP/mL/h and from 13.70 to 118.32 nmol pNP/mL/h, respectively. At the end of the experiment, a decrease in the acid (82.09 nmol pNP/mL/h) and alkaline (98.17 nmol pNP/mL/h) phosphatase activity was recorded. No significant differences were registered between the activities of the two phosphatases during the entire incubation period—Figure 4b.

3.5. Effect of Abiotic Stress Conditions on Bacillus safensis P1.5S P Solubilization Ability

The P solubilization potential of B. safensis P1.5S was evaluated using abiotic stress inducers such as temperature, alkalinity and salinity. The tested strain was able to solubilize TCP from the PVK medium regardless of the stress conditions, with slight differences depending on the tested parameters. Thus, the P1.5S strain optimally solubilized P during the incubation at 20 °C, with a gradual accumulation of soluble phosphorus up to 7 days, when the maximum amount (19.54 µg/mL) was recorded—Figure 5(a1). When 37 °C was used as the incubation temperature, the P1.5S strain solubilized phosphorus with values ranging from 14.39 to 16.21 µg P/mL. In relation to the control (28 °C), an increase of P solubilization was registered at 20 °C in samples taken at 5 and 7 DAI. No significant differences were observed at the beginning (3 DAI) and at the end (10 DAI) of the incubation period between solubilized P amounts determined at different incubation temperatures, except for those recorded at 20 °C and 37 °C at 10 DAI. In addition to P solubilization, the growth of the P1.5S strain was also assessed during the incubation period (Figure 5(a2)). The highest growth (8.92 log10 CFU/mL) was recorded for the P1.5S culture incubated at 20 °C. Incubation at 37 °C resulted in a reduced growth compared with both control and 20 °C, with the lowest number of viable cells present in the PVK medium at 10 DAI (7.20 log10 CFU/mL).
Cultivation in alkaline conditions (pH 9) resulted in minor increases of P solubilization compared with the control (pH 7)—Figure 5(b1). Soluble P gradually accumulated in the PVK medium initially adjusted to pH 9 until day 7, followed by a significant decrease at 10 DAI (p < 0.0001). Amounts of solubilized P ranged between 14.69 and 18.73 µg P/mL during the entire incubation period. Regarding the bacterial growth, with the exception at 7 DAI, no significant differences were observed between the number of CFU/mL determined in the PVK medium with the initial pH of 7 or 9. The maximum number of cells was detected in the control 10 days after inoculation (8.20 log10 CFU/mL)—Figure 5(b2).
Salinity did not influence the ability of Bacillus safensis P1.5S to solubilize TCP from Pikovskaya’s broth, as Figure 5(c1) shows. The amount of soluble P ranged between 14.72 and 16.89 µg P/mL, with no significant differences recorded between control (0.150 g/L) and different NaCl concentrations (2.5 g/L, 10 g/L and 15 g/L). However, increasing concentrations of NaCl stimulated growth at 3, 5 and 7 DAI, suggesting that the P1.5S strain exhibits tolerance to salinity—Figure 5(c2).
P solubilization under different abiotic stress conditions was accompanied by a decrease in the medium’s pH compared to the uninoculated PVK, as shown in Figure 5(a,b,c3).

3.6. Bacillus safensis P1.5S Displayed a Distinct Pattern of Organic Acid Production Under Stress Conditions

HPLC analysis of B. safensis P1.5S supernatants confirmed the production and release of organic acids such as lactic, acetic, succinic, citric, malic/formic, oxalic, tartaric/gluconic and an unknown acid during P solubilization under stress conditions. Temperature, alkalinity and salinity used as abiotic stress inducers influenced organic acid production. Thus, their number, release time and concentration varied depending on the tested conditions.
Organic acid production was influenced by different incubation temperatures. Lactic acid was evidenced in the samples taken at 3, 5 and 7 DAI from the cultures incubated at 20 °C and 37 °C and it was not found at 10 DAI—Figure 6a,b. The release timing was similar with the control (28 °C), but lower concentrations were recorded during the incubation at 20 °C (3.79 mM) and 37 °C (6.24 mM) compared with 28 °C (8.03 mM). Additionally, the release pattern differed during the 20 °C incubation of the P1.5S strain, with lactic acid production showing no significant differences at 3, 5, and 7 DAI (p > 0.05). Unlike the control, where lactic acid production increased up to 5 DAI (8.03 mM) before significantly decreasing to 7 DAI (3.17 mM, p < 0.0001), a gradual decrease was observed from 3 DAI (6.24 mM) when incubation was conducted at 37 °C (2.76 mM at 7 DAI). Acetic acid was evidenced in all samples from cultures incubated at 20 °C and 37 °C, in contrast with the control, in which acetic acid was not detected at 5 DAI—Figure 6a,b. Except for this difference, the release pattern was quite similar, but it should be emphasized that in the cultures incubated at 37 °C higher concentrations were detected at 7 and 10 DAI (10.05 mM and 15.05 mM, respectively) compared to the control. Also, lower acetic acid concentrations were observed during the entire cultivation period at 20 °C versus control. The succinic acid release pattern was not significantly different compared to the control in relation to temperature changes, a gradual accumulation being recorded across all samples and the control during the incubation period. However, our data indicates that incubation at 28 °C stimulated succinic acid production (9.18 mM), yielding higher concentrations compared to 20 °C (7.74 mM) and 37 °C (4.10 mM). A modification in citric acid production was observed in the P1.5S cultures incubated at 20 °C and 37 °C. In these temperature conditions, citric acid was released into the culture medium throughout the entire incubation period, whereas it was detected only at 7 and 10 DAI in the control. A gradual increase in citric acid production was observed across all samples and the control, with the highest concentration of citric acid (1.161 mM) recorded at 10 DAI in the P1.5S culture incubated at 37 °C. Except at 5 DAI, no significant differences in malic/formic acid production and release time (p > 0.05) were evidenced between the samples and control—Figure 6a,b.
Depending on the sampling moment, alkaline conditions influenced the number of organic acids produced by the P1.5S strain, stimulating the synthesis of oxalic, tartaric/gluconic, acetic, and succinic acids compared with the pH 7 control—Figure 6c. An exception was observed at 3 DAI, when lactic, acetic, succinic, oxalic, and malic/formic acids were detected both in the sample (initial pH 9) and control (initial pH 7). Oxalic, lactic, and succinic acids were produced regardless of the initial pH at 5 DAI, with no significant differences in their concentrations. However, malic/formic and tartaric/gluconic acids were detected only in control, whereas citric and acetic acids were identified exclusively in the PVK broth with an initial pH of 9. At 7 DAI, the same organic acids were released irrespective of the initial pH value tested. Quantitative differences were observed only for oxalic and succinic acids, which were produced in higher concentrations at pH 9 (0.127 mM and 8.68 mM, respectively). At the end of the incubation period (10 DAI), HPLC analysis of the supernatants taken from PVK with an initial pH of 9 revealed increased concentrations of acetic (14.52 mM), succinic (13.01 mM), and oxalic (0.209 mM) acids compared with the control. In addition, lactic acid was detected only in the B. safensis P1.5S culture incubated in the medium with an initial pH of 9—Figure 6c.
The comparative analysis of the HPLC chromatograms revealed that the release of organic acids varied depending on the NaCl concentrations used to supplement the PVK medium (Figure 6d–f). At the beginning of the incubation period (3 DAI), the three major organic acids (lactic, acetic, and succinic) were produced at all tested salt concentrations. However, saline stress induced a different release pattern. Thus, B. safensis P1.5S produced more acetic acid under salt stress compared to the control (0.150 g/L NaCl), where lactic acid was released as the predominant acid. Additionally, citric acid was detected only at the highest tested NaCl concentration (15 g/L), while tartaric/gluconic and malic/formic acids were not identified at this concentration. Oxalic acid was detected in the PVK broth supplemented with various NaCl concentrations, except at 10 g/L NaCl. At 5 DAI, lactic acid production was found to decrease as the NaCl concentration in the PVK medium increased. Similar to the control, acetic acid was not detected at 10 g/L NaCl, but it was present at 2.5 and 15 g/L NaCl (6.03 mM and 4.72 mM, respectively). Salinity inhibited the production of succinic and oxalic acids but stimulated the release of tartaric/gluconic acids, with a higher concentration at 10 g/L NaCl (0.432 mM). Citric, lactic and succinic acids were synthesized irrespective of the NaCl concentration, as shown by the 7 DAI samples. Acetic and oxalic acids were not detected in the PVK medium supplemented with 10 g/L NaCl, although both acids were present in all other samples. At the end of the incubation period (10 DAI), increased salt concentrations inhibited the production of oxalic, malic/formic, citric, and acetic acids, but stimulated the release of tartaric/gluconic acid (especially at 10 g/L NaCl). At 15 g/L, neither tartaric/gluconic acid nor malic/formic acid were detected.

3.7. Temperature Induces Changes in Acid and Alkaline Phosphatase Activity During Phosphorus Solubilization

The results depicted in Figure 7a,b show that incubation temperature significantly influenced the activity of acid and alkaline phosphatases produced by B. safensis P1.5S. Thus, at multiple sampling times, the activity of both enzymes increased at 20 °C and 37 °C compared to the control (28 °C). The maximum activity (180.456 nmoles pNP/mL/h for acid phosphatase and 157.867 nmoles pNP/mL/h for alkaline phosphatase) was recorded at 10 DAI in cultures incubated at 20 °C. An increase in acid phosphatase activity was induced by the incubation at 20 °C at 3 and 10 DAI, while growth at higher temperatures (37 °C) resulted in increased activity at 5 DAI (Figure 7(a1)). No significant differences were observed between the incubation temperatures at 7 DAI for acid phosphatases. Significantly higher alkaline phosphatase activities were recorded when the P1.5S strain was exposed to 37 °C at 3 and 5 DAI, compared with the control (Figure 7(b1)).
Alkalinity did not change significantly the dynamic of acid and alkaline phosphatase production during B. safensis P1.5S cultivation in the PVK medium with the initial pH adjusted to 9. Enzyme activity increased exponentially until the seventh DAI, when the maximum value was recorded (114.07 nmoles pNP/mL/h and 96.70 nmoles pNP/mL/h, respectively), followed by a decrease at 10 DAI (Figure 7(a2,b2)). The activity of both enzymes slightly increased at pH 9 at 3 and 5 DAI compared with the control (pH 7).
Salinity did not affect acid and alkaline phosphatase activity during the ten-day incubation period, as shown in Figure 7(a3,b3). The only significant difference was recorded for the acid phosphatase at 5 DAI, when higher NaCl concentrations inhibited enzymatic activity—Figure 7(a3).

4. Discussion

Climate change represents a major threat to crop yields and food production by altering global and regional rainfall and temperature patterns, affecting soil fertility and reducing the availability of nutrients for plant growth [39]. Phosphorus is one of the most important macronutrients for plant growth influenced by abiotic stressors. While P chemical fertilizers are easily accessible to plants, their application to soils faces challenges like poor diffusion, limited solubility, and fixation to mineral surfaces, increasing the amount of P that becomes unavailable to plants [9]. A sustainable alternative to chemical fertilizers is the use of phosphate-solubilizing bacteria that tolerate abiotic stress [40].
In this study, we isolated a novel Bacillus safensis strain with P-solubilizing capabilities. Previous studies reported different B. safensis strains as tolerant to abiotic stressors [18,23,41]. However, the influence of abiotic stress on the phosphorus-solubilizing capacity of B. safensis remains poorly understood. Therefore, we used the B. safensis P1.5S strain to investigate the mechanisms underlying tricalcium phosphate solubilization under different stress conditions. The first step was to assess its tolerance to the abiotic stress induced by variations in pH, temperature, and salinity. Bioinformatic analysis revealed that the B. safensis P1.5S genome contains multiple genes associated with tolerance to pH, temperature, salinity and drought, which are responsible for bacterial survival in harsh environments (Table S2). One example is the sigB gene responsible for the transcription of approximately 200 genes encoding proteins involved in stress tolerance [42]. Some of the stress response genes identified in the P1.5S draft genome, such as heat shock proteins groEL and dnaK, may be essential for PSB exposed to hostile conditions. Moreover, according to the genomic analysis, to maintain the cellular and functional integrity of the bacterium in stressful conditions, B. safensis P1.5S could produce osmolytes (Table S3) and antioxidant enzymes (Table S4) like superoxide dismutase (sodA). Osmolytes provide osmotic balance and protein stability, while antioxidant enzymes ameliorate the effects of reactive oxygen species, ensuring bacterial survival [43]. Genes involved in proline (proA, proB, proH) and glutamate biosynthesis (gltA, gltB, gltT), related to glycine betaine metabolism (opuAB), as well as those responsible for trehalose transport (treA, treP) are associated with osmotic response in bacteria [44]. Organic acid excretion during phosphorus solubilization leads to increased osmolality, which may be mitigated by the secretion of proline and glutamate, along with the accumulation of glycine betaine and trehalose [45].
To confirm the stress tolerance of the P1.5S strain, growth experiments were employed in LB medium using different temperatures (20 °C, 28 °C, 37 °C), pH (5, 7, 9), and salinity (2.5 g/L, 10 g/L, 15 g/L) conditions. Our results indicate that B. safensis P1.5S can grow under various stressful conditions, suggesting that the strain is tolerant to temperature, pH, and salt stress (Figure 2). However, different conditions influence bacterial growth. Thus, incubation at 37 °C significantly enhanced B. safensis P1.5S growth compared to the control throughout the experimental period, indicating its potential to grow under elevated temperatures. Heat-tolerance was previously reported for other B. safensis strains, such as for SCAL1, capable of growing at 32, 50, and 60 °C, supporting the possibility of using B. safensis as a solution to mitigate heat stress [18]. Lower incubation temperatures (20 °C) were associated with a prolonged lag phase (up to 6 h) and decreased growth compared with the control. However, after 24 h of incubation, no significant differences were recorded versus the control, supporting the hypothesis that B. safensis P1.5S is temperature-tolerant. B. safensis P1.5S is able to grow in LB medium with an initial pH of 5 and 9. However, exposure to acidic and alkaline conditions after inoculation resulted in a growth delay of approximately 4 h, with lower OD600 values recorded up to 24 h compared with the control (pH 7). Although no significant differences were recorded up to 12 h, the cultivation at pH 5 showed better growth after 24 h of incubation compared to pH 9 cultures. The tolerance of Bacillus safensis P1.5S at pH 5 may be a key factor for its survival in soils affected by acidification and reduced precipitation [46]. The different salt concentrations used to supplement the LB broth did not significantly influence the growth of strain P1.5S during the incubation period, suggesting its capability to grow in highly saline soils, characterized by a salt concentration higher than 10.024 g/L [47]. The salt tolerance of B. safensis was also reported for other strains like B. safensis PM22 [23] or B. safensis P-14 [48], considered by the authors as halotolerant.
B. safensis P1.5S is a bacterium with P solubilization potential, as revealed by the TCP solubilization assay. No significant differences were observed between sampling times, suggesting that P1.5S maintains the capacity to solubilize TCP throughout the incubation period (Figure 3). Lower or similar amounts of soluble phosphorus have been previously reported for other Bacillus strains. Thus, [49] reported that Bacillus subtilis IA6, isolated from cotton fields, exhibited a lower capacity to solubilize TCP (2.38 µg/mL) compared to our strain. A similar solubilization ability was observed for several Bacillus cereus (9.5–17.5 µg/mL) and Bacillus megaterium (4.4–26.5 µg/mL) strains isolated from paddy fields [50]. Additionally, another study [51] showed that the Bacillus thuringiensis GS1a strain, isolated from arid soils, solubilized approximately 20 mg/L of TCP. Comparable results were reported for Bacillus sp. PVMX4 and B. megaterium MTCC2444, which solubilized 32.1 µg/mL and 29.2 µg/mL, respectively [52]. Furthermore, the authors of [53] demonstrated that B. vallismortis 20P and B. tequilensis 28P strains solubilized comparable amounts of phosphorus (20.43 µg/mL and 28.42 µg/mL), similar to the amounts recorded for the B. safensis P1.5S strain. Other B. safensis strains, like P-14 (115.89 µg/mL) [48] and BaT-68s (114.24 mg/L) [54], have demonstrated enhanced phosphate-solubilizing potential, highlighting the efficacy of B. safensis as a phosphate-solubilizing bacterium. Data analysis revealed that the accumulation of soluble P in the PVK broth is related to a pH decrease. The reduction of medium pH in relation to P solubilization has been previously reported [55,56,57], being corelated with the production and release of organic acids—one of the most important mechanisms involved in inorganic P solubilization [58,59]. BLAST analysis of the B. safensis P1.5S genome revealed at least 32 genes involved in the biosynthesis of organic acids associated with the solubilization of insoluble P compounds, including gluconic, formic, malic, citric, lactic, acetic, and succinic acids (Table S1). The predicted organic acids are consistent with those reported in previous studies investigating the mechanisms of P solubilization [55,60,61]. Several genes known to play an essential role in the regulation of the P solubilization process were identified in the P1.5S genome, including the following: kduD (gluconate-2-dehydrogenase), gdh (glucose-1-dehydrogenase), gcd (quinoprotein glucose-1-dehydrogenase), poxB (pyruvate dehydrogenase) and ldh (lactate dehydrogenase) [62,63]. However, no genes encoding the pqqA-E operon, which is involved in gluconic acid synthesis [64,65], were identified in the genome of Bacillus safensis P1.5S, suggesting that the production of this acid may be encoded by different genes.
The HPLC analysis of B. safensis P1.5S culture supernatants confirmed the production and release of organic acids, these results supporting the phosphorus solubilization ability of B. safensis P1.5S (Figure 4a). Considering the pKa values, the most potent acids and, therefore, the more efficient ones in solubilizing TCP (oxalic, tartaric/gluconic, malic/formic and citric acids) were synthesized in lower concentrations, whereas weaker acids such as lactic, acetic, and succinic acids were produced in higher amounts by B. safensis P1.5S. Organic acids produced by PSB can act in several ways to dissociate insoluble phosphorus compounds, causing a decrease in soil pH, and chelating cations such as calcium, iron and aluminum that are bound to the phosphate group via carboxyl and hydroxyl groups. Furthermore, organic acids can compete with phosphate ions for adsorption sites in the soil, preventing the adsorption and immobilization of phosphate, thus keeping it in a form available to plants [66,67].
A literature review on organic acid production during TCP solubilization by various bacteria revealed that some of the organic acids produced by Bacillus safensis P1.5S (lactic, acetic, and succinic acids) were also detected in the supernatant of Bacillus strains isolated from wheat roots and the rhizosphere [68]. It was also observed that the strain P1.5S produced higher concentrations of lactic, acetic, and succinic acids compared to B. megaterium ZR32, B. megaterium ZR19, B. subtilis ZE15, and B. subtilis ZE3 strains. Although the concentrations of organic acids produced by the P1.5S strain were higher, the B. megaterium and B. subtilis strains solubilized significantly higher amounts of phosphorus (40–130 µg/mL) compared to B. safensis P1.5S.
Another mechanism involved in inorganic P solubilization is the dephosphorylation of molecules mediated by acid and alkaline phosphatases [58]. Several genes involved in the synthesis of phosphatases were evidenced in the B. safensis P1.5S genome, including phoA, one of the three homologous genes (phoA, phoD, phoX) encoding alkaline phosphatases. Although phoD and phoX are the most frequently identified genes when bacteria are incubated in inorganic P-deficient media [12], our analyses did not reveal their presence in the P1.5S genome. However, the ycsE and yitU genes, involved in phosphatase production, were identified. Also predicted in the P1.5S genome were genes responsible for regulating the response to P starvation, such as the two-component regulatory system encoded by phoB and phoR, and the phoP gene which encodes a transcriptional protein that regulates alkaline phosphatase synthesis [69]. Bacteria with phosphorus-solubilizing potential can also produce acid phosphatases encoded by the phoC and acpA genes [69], but only the acpA gene was predicted for the B. safensis P1.5S strain. The analysis of the culture supernatants showed that higher activities were recorded for both acid and alkaline phosphatases at the end of the incubation period (7 and 10 DAI), with no significant differences between the two enzymes (Figure 4b). Statistical analysis revealed a strong positive correlation between the amount of solubilized phosphorus and the activity of acid (r = 0.7757, p = 0.2243) and alkaline phosphatases (r = 0.7399, p = 0.2601), supporting their potential involvement in the ability of B. safensis P1.5S to solubilize TCP. The contribution of acid and alkaline phosphatases in P solubilization was previously shown for other B safensis strains [70], as well as for other Bacillus species such as B. flexus, B. megaterium [71], B. mycoides and B. pumilus [72].
A further step in our investigation was to assess the P solubilization capability of B. safensis P1.5S under abiotic stress conditions. The phosphorus solubilization potential of the B. safensis P1.5S strain was not affected by abiotic stressors, including temperature, alkalinity, and salinity. However, some variations were registered, depending on the tested conditions.
Temperature plays a crucial role in bacterial P solubilization by affecting microbial growth, enzyme activity, and metabolic processes [73]. Our data analysis revealed that the amount of soluble P detected in the PVK broth did not change significantly at the beginning and the end of the experiments, regardless of the selected temperature (Figure 5a). Except for at 5 DAI, incubation at 37 °C resulted in non-significant differences of soluble P amounts compared with the control (28 °C). A minor increase in P solubilization was observed at 5 and 7 DAI in the culture incubated at 20 °C, in a direct relationship with bacterial growth, as indicated by Pearson’s correlation analysis (r = 0.9925, p = 0.0008).
Concerning the influence of pH, only alkaline conditions could be tested in our study. The effect of low pH on the solubilization potential of the P1.5S strain could not be evaluated, as phosphorus solubilization occurred in both the uninoculated PVK broth and inoculated medium, most likely due to acidic conditions. Usually, increasing soil alkalinity can reduce the effectiveness of microbial P solubilization [74]. Our data showed that alkalinity slightly increased the P amount detected in the culture medium at 5 and 7 DAI, with no significant differences at 3 and 10 DAI compared with the control (pH 7)—Figure 5b. Additionally, no significant effect of pH 9 on bacterial growth was observed, suggesting that B. safensis P1.5S has the potential to survive and solubilize phosphorus under alkaline stress.
High salt concentrations in soil reduce P solubilization, mostly by decreasing the activity of enzymes such as the acid and alkaline phosphatases which are involved in P solubilization [75]. B. safensis P1.5S was able to solubilize P regardless of the NaCl concentrations used to supplement the PVK broth, with no significant differences recorded throughout the entire incubation period—Figure 5c. Moreover, the salinity conditions used in our experiments did not affect the acid and alkaline phosphatase activity, as shown in Figure 7, highlighting the tolerance of B. safensis P1.5S. Compared to Bacillus strains which were first isolated from peanut [33], our results revealed that the B. safensis P1.5S strain is more efficient in solubilizing TCP under salinity, pH, and temperature stress conditions. The results presented by [76] showed a negative effect of pH 9 on the solubilization potential, contrary to the data presented in this study for B. safensis P1.5S. On the other hand, experiments performed by [77] showed that B. safensis AL isolated from halophilic plants solubilized significantly higher amounts of TCP (80 and 500 µg/mL) at 2.337 g/L, 9.350 g/L, and 18.701 g/L NaCl, compared with B. safensis P1.5S in similar conditions.
Abiotic stress conditions affected the organic acid production pattern during TCP solubilization, inducing both quantitative and qualitative changes. The number of secreted organic acids, their release time and concentration varied depending on the stress condition and sampling time. Some organic acids, such as acetic acid, were produced at higher concentrations versus control, while others (e.g., lactic and succinic acids) were released in smaller amounts, depending on the temperature (Figure 6a,b). Incubation at 37 °C slightly increased the concentrations of some organic acids released in the PVK broth, while minor decreases were recorded at 20 °C. At specific incubation times, acetic and citric acid were detected only in cultures incubated at 20 °C and 37 °C. Alkaline conditions stimulated the production of some acids (oxalic and succinic acids), while inhibiting the synthesis of others (malic/formic and tartaric/gluconic acids) at specific sampling times (Figure 6c). Salt stress stimulated the production of acetic and citric acids, while other acids such as tartaric/gluconic and malic/formic acids were not detected at higher NaCl concentrations (Figure 6d–f). However, it is important to emphasize that despite different organic acid production patterns induced by abiotic stressors, the P solubilization potential of B. safensis P1.5S was not significantly altered under abiotic stress.
The production of acid and alkaline phosphatases by B. safensis P1.5S was not significantly influenced by alkaline and salt stress and showed minor increases at 20 °C and 37 °C compared with the control (28 °C). The two enzymes showed activity under all tested stress conditions, with maximum values recorded after cultivating B. safensis P1.5S at 20 °C. Statistical analysis suggests that phosphatases produced at 20 °C and 2.5, 10 or 15 g/L NaCl were not involved in TCP solubilization since the correlations established between the amounts of soluble phosphorus and the enzymatic activities in the PVK medium were weak (r = 0.2449–0.4828, p = 0.7551–0.4100). On the other hand, a strong direct correlation between the activity of the two phosphatases and the amounts of phosphorus solubilized at pH 9 was shown by the Pearson correlation coefficient (acid phosphatase: r = 0.7144, p = 0.1751; alkaline phosphatase: r = 0.7664, p = 0.1307), suggesting the involvement of the two enzymes in the solubilization process. At 37 °C, a strong inverse correlation was found between the activity of the acid (r = −0.8341, p = 0.1659) and alkaline phosphatases (r = −0.7846, p = 0.2154) and the amounts of phosphorus solubilized. Although the Pearson correlation coefficient suggested a relationship between the two variables in some cases, the correlation was not statistically significant (p > 0.05), indicating that the association may be purely coincidental. Similar observations showing the lack of correlation between the amounts of solubilized P and the ability of the two enzymes to dissociate the phosphorus source were previously reported [78]. Based on these results, we infer that the primary mechanism employed by B. safensis P1.5S for TCP solubilization is the production of organic acids. However, at certain sampling points, the acid production alone cannot fully explain the solubilization of TCP. Therefore, we do not exclude the possibility of an additional mechanism acting synergistically with the organic acids to dissolve the insoluble phosphorus source.

5. Conclusions

This study shows that Bacillus safensis P1.5S is a phosphate-solubilizing bacterium tolerant to temperature, pH, and salt stress. The strain demonstrated the ability to grow at elevated temperatures (37 °C), in high NaCl concentrations (15 g/L), and in both alkaline and acidic conditions, suggesting its potential to survive in challenging environments. The production of organic acids, including lactic, acetic, and succinic acid, appears to be the primary mechanism involved in P solubilization, although the results also support the potential involvement of both acid and alkaline phosphatases. Different abiotic stress conditions influenced the pattern of organic acid production, but did not significantly affect the efficacy of strain P1.5S to solubilize inorganic phosphates (19.54 µg/mL). Our results indicate B. safensis P1.5S as a promising tool for enhancing phosphorus availability to plants by mobilizing the soil P reserves. Its application could be beneficial in agriculture, especially in regions facing soil degradation due to high salinity, alkalinity or temperatures. However, further studies are needed to fully assess the strain’s potential as a biofertilizer. Future research should focus on investigating the long-term effect of abiotic stressors on the viability and efficiency of the P1.5S strain, exploring its interactions with various soil microbial populations, evaluating the strain’s performance under field conditions, and assessing the environmental risks associated with its introduction into agricultural ecosystems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11040388/s1, Table S1. Genes involved in organic acids synthesis putatively encoded by the Bacillus safensis P1.5S genome; Table S2. Genes involved in pH, temperature, salinity or drought tolerance putatively encoded by the Bacillus safensis P1.5S genome; Table S3. Genes involved in osmolyte production putatively encoded by the Bacillus safensis P1.5S genome; Table S4. Genes involved in oxidative stress tolerance putatively encoded by the Bacillus safensis P1.5S genome.

Author Contributions

Conceptualization, M.S.; methodology, M.S. and M.M.; software, M.M. and A.E.-S.; validation, M.S. and M.M.; formal analysis, L.-E.M. and A.E.-S.; investigation, L.-E.M.; resources, M.S. and M.M.; data curation, A.E.-S. and L.-E.M.; writing—original draft preparation, L.-E.M.; writing—review and editing, M.S., M.M. and A.E.-S.; visualization, M.S.; supervision, M.S. and M.M.; project administration, M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data for the draft genome of Bacillus safensis P1.5S are available under BioProject PRJNA960951, Assembly GCA_029930515.1, and GenBank accession JARZFW000000000. All other datasets are available upon request from the authors.

Acknowledgments

The authors would like to thank Denis Constantin Topa of Iasi University of Life Sciences, Romania for his support in collecting soil samples.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PSMPhosphorus-Solubilizing Microorganisms
PSBPhosphorus-Solubilizing Bacteria
PPhosphorus
LBLuria Bertani agar/broth
PVKPikovskaya agar/broth
RASTRapid Annotations using Subsystems Technology
BLASTBasic Local Alignment Search Tool
HPLCHigh-Performance Liquid Chromatography
TYGSType (Strain) Genome Server
ODOptical Density
TCPTri-Calcium Phosphate
CFUColony Forming Units
DAIDays After Inoculation
pNPρ-Nitrophenol
SEMStandard Error of the Mean
DNADeoxyribonucleic Acid
RNARibonucleic acid
dDDHDigital DNA-DNA Hybridization

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Figure 1. The RAST analysis-based subsystem distribution of the genome sequence of Bacillus safensis strain P1.5S. Each color in the pie graph represents a particular group of genes mentioned on the right side of the graph.
Figure 1. The RAST analysis-based subsystem distribution of the genome sequence of Bacillus safensis strain P1.5S. Each color in the pie graph represents a particular group of genes mentioned on the right side of the graph.
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Figure 2. Effect of temperature (a), pH (b) and different NaCl concentrations (c) on Bacillus safensis P1.5S growth during 72 h of incubation. The bacterial cells were cultivated in LB medium. Values represent the mean ± standard error of the mean.
Figure 2. Effect of temperature (a), pH (b) and different NaCl concentrations (c) on Bacillus safensis P1.5S growth during 72 h of incubation. The bacterial cells were cultivated in LB medium. Values represent the mean ± standard error of the mean.
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Figure 3. Dynamics of phosphorous solubilization, pH and viable bacterial cells in PVK medium during the 10 days of incubation. Bacterial cells were cultured in PVK medium with tricalcium phosphate as the insoluble P source and incubated at 28 °C. Values represent the mean ± standard error of the mean. DAI—days after inoculation.
Figure 3. Dynamics of phosphorous solubilization, pH and viable bacterial cells in PVK medium during the 10 days of incubation. Bacterial cells were cultured in PVK medium with tricalcium phosphate as the insoluble P source and incubated at 28 °C. Values represent the mean ± standard error of the mean. DAI—days after inoculation.
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Figure 4. Bacillus safensis P1.5S produced organic acids (a) and phosphatases (b) during tricalcium phosphate solubilization in PVK medium at 28 °C. Values represent the mean ± standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). ns—no significant differences. DAI—days after inoculation.
Figure 4. Bacillus safensis P1.5S produced organic acids (a) and phosphatases (b) during tricalcium phosphate solubilization in PVK medium at 28 °C. Values represent the mean ± standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). ns—no significant differences. DAI—days after inoculation.
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Figure 5. Effect of temperature (a), alkalinity (b), and salinity (c) on Bacillus safensis P1.5S P solubilization (1), bacterial growth (2), and pH variation (3) during 10 days of cultivation in PVK medium. Control in panel (a3) = uninoculated PVK incubated at 20 °C, 28 °C and 37 °C; control pH 7 and control pH 9 in panel (b3) = uninoculated PVK with pH initially adjusted to 7 and 9; control in panel (c3) = uninoculated PVK supplemented with 0.150 g/L, 2.5 g/L, 10 g/L and 15 g/L NaCl. Values represent the mean ± the standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). ns—no significant differences. DAI—days after inoculation.
Figure 5. Effect of temperature (a), alkalinity (b), and salinity (c) on Bacillus safensis P1.5S P solubilization (1), bacterial growth (2), and pH variation (3) during 10 days of cultivation in PVK medium. Control in panel (a3) = uninoculated PVK incubated at 20 °C, 28 °C and 37 °C; control pH 7 and control pH 9 in panel (b3) = uninoculated PVK with pH initially adjusted to 7 and 9; control in panel (c3) = uninoculated PVK supplemented with 0.150 g/L, 2.5 g/L, 10 g/L and 15 g/L NaCl. Values represent the mean ± the standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). ns—no significant differences. DAI—days after inoculation.
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Figure 6. Effect of temperature (a,b), alkalinity (c), and salinity (d–f) on the organic acids production by Bacillus safensis P1.5S during 10 days of cultivation in PVK medium. Values represent the mean ± the standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). DAI—days after inoculation.
Figure 6. Effect of temperature (a,b), alkalinity (c), and salinity (d–f) on the organic acids production by Bacillus safensis P1.5S during 10 days of cultivation in PVK medium. Values represent the mean ± the standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). DAI—days after inoculation.
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Figure 7. Effect of temperature (1), alkalinity (2) and salinity (3) on acid (a) and alkaline (b) phosphatases during the 10 days of cultivation of Bacillus safensis P1.5S in PVK medium. Values represent the mean ± the standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). ns—no significant differences. DAI—days after inoculation.
Figure 7. Effect of temperature (1), alkalinity (2) and salinity (3) on acid (a) and alkaline (b) phosphatases during the 10 days of cultivation of Bacillus safensis P1.5S in PVK medium. Values represent the mean ± the standard error of the mean. Asterisk marks significant differences (* = p ˂ 0.05, ** = p ˂ 0.01, *** = p ˂ 0.001; **** = p < 0.0001). ns—no significant differences. DAI—days after inoculation.
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MDPI and ACS Style

Mantea, L.-E.; El-Sabeh, A.; Mihasan, M.; Stefan, M. Bacillus safensis P1.5S Exhibits Phosphorus-Solubilizing Activity Under Abiotic Stress. Horticulturae 2025, 11, 388. https://doi.org/10.3390/horticulturae11040388

AMA Style

Mantea L-E, El-Sabeh A, Mihasan M, Stefan M. Bacillus safensis P1.5S Exhibits Phosphorus-Solubilizing Activity Under Abiotic Stress. Horticulturae. 2025; 11(4):388. https://doi.org/10.3390/horticulturae11040388

Chicago/Turabian Style

Mantea, Loredana-Elena, Amada El-Sabeh, Marius Mihasan, and Marius Stefan. 2025. "Bacillus safensis P1.5S Exhibits Phosphorus-Solubilizing Activity Under Abiotic Stress" Horticulturae 11, no. 4: 388. https://doi.org/10.3390/horticulturae11040388

APA Style

Mantea, L.-E., El-Sabeh, A., Mihasan, M., & Stefan, M. (2025). Bacillus safensis P1.5S Exhibits Phosphorus-Solubilizing Activity Under Abiotic Stress. Horticulturae, 11(4), 388. https://doi.org/10.3390/horticulturae11040388

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