1. Introduction
Phosphorus (P) is one of the major macronutrients for plant growth [
1]. Therefore, in intensive agriculture, a supply of phosphorus in the form of fertilizer is crucial to obtaining good yields. As the reserves of degradable natural phosphorus are limited, a targeted and environmentally friendly supply is necessary for agriculture.
P is present in a range of organic and inorganic forms in soils. Inorganic P is typically found in soil as insoluble mineral compounds, some of which develop after continuous chemical fertilizer treatments [
2,
3]. However for plants, soil phosphorus is generally accumulated in chemical forms which are unavailable to them [
4]. It usually takes the form of a phosphate ion, which is a charged ion that is bonded to other components to make a molecule. Such insoluble compounds comprise calcium phosphate in alkaline soils, and iron phosphate, and aluminum phosphate in acidic soils. Referring to Zou et al. [
5], only ≈0.1% of the total P reserve in the soil is in the soluble form and available for plant uptake; generally, calcium phosphate is more soluble than aluminum phosphate, while the latter is more soluble than iron phosphate [
6]. Most of the phosphates in the soil are absorbed into soil particles or incorporated into soil organic matter [
7]. As well, a great portion of the soluble phosphate applied to the soil as chemical fertilizer is immobilized rapidly and becomes unavailable to plants [
1,
8].
Soil microorganisms like bacteria, fungus, and actinomycetes are involved in a variety of processes that lead to the transformation of soil P and are thus an integral part of the soil P cycle. In particular, soil microorganisms are efficient in releasing and unblocking inorganic and organic P from total soil P via solubilization and mineralization [
9]. Currently, the primary goal of soil phosphorus management is to maximize crop yield while minimizing P loss from soils. The ability of a PSB to convert insoluble forms of phosphorus to accessible forms is an important trait in plant growth-promoting bacteria for increasing plant yields, and the use of PSB as inoculants to increase plant P uptake has increased [
1,
10].
In recent years, various studies have presented a large number of new PSB [
10,
11,
12,
13,
14,
15]. A great percentage of those were preliminary studies, conducted only in vitro and without plant and field application. Most of these studies assume that in vitro P solubilization ability will translate into available P for plant uptake in the soil.
To assess bacteria’s ability to dissolve insoluble phosphates, solid and liquid media techniques are used, and are based on media with a source of phosphorus unavailable to the bacteria [
6]. The solid medium gives qualitative solubilization efficiency; the solubilization index is estimated by measuring the growing diameter of the colony and the solubilization halo in plates [
16]. Moreover, the liquid medium gives quantitative solubilization efficiency through determining the pH changes and the soluble phosphate concentration in the medium [
17]. Although most researchers up to now have relied on calcium phosphate as a universal source of phosphate to determine and judge that the bacteria tested are phosphate solubilizers, the authors of [
8] recommended that the sole use of this phosphate to identify soil microorganisms as potential P solubilizers is not sufficient and that aluminum and iron phosphates should be tested as well.
Phosphate solid sludge is a byproduct of the phosphate extraction industry’s exploitation and subsequent metallurgical treatment. The processing of phosphates creates a lot of sludge, which accumulates, forms fillings, reduces arable land, and changes the landscape. The phosphate sludge is mainly composed of phosphorus, minerals, and some of the original pollutants. To obtain some of these minerals, such as phosphorus, we thought about using this sludge as an agricultural substrate. One of the stages of this project was to isolate PSB from this sludge in order to valorize it as a biofertilizer. With the emphasis on isolation and screening of potential PSB for agricultural aims, this study was performed to evaluate the biochemical and genetic characteristics of PSB isolated from the phosphate solid sludge and to evaluate their capacity to solubilize three forms of phosphates unavailable to plants: calcium phosphate (Ca3(PO4)2), aluminum phosphate (AlPO4), and iron phosphate (FePO4).
2. Materials and Methods
2.1. Isolation and Screening of Phosphate Solubilizing Bacteria
Phosphate solid sludge samples were collected from the phosphate mining center of Khouribga (Morocco; 32°45′17.7645″, 006°51′14.5182″); One gram of each phosphate solid sludge sample was added to 9 mL of phosphate buffer saline (pH 7.2), serial dilutions from 10
−1 up to 10
−6 were realized. Then, 100 µL of 10
−3 to 10
−6 serially suspension was spread on NBRIP solid medium (10 g/L
d-glucose, 5 g/L magnesium chloride hexahydrate, 0.25 g/L magnesium sulfate heptahydrate, 0.2 g/L potassium chloride, 0.1 g/L ammonium sulfate, amended with 5.0 g/L tricalcium phosphate (Ca
3(PO
4)
2) as a sole source of P [
18]. Then the Petri dishes were incubated at 30 °C for 5 days. The bacterial colonies with a clear halo zone were selected and purified three times on NBRIP solid medium. The qualitative efficiency of the selected PSBs was tested according to the solubilization index, measured by the formula PSI = C + H/C, (C = Colony diameter; H = Halo zone diameter) [
16]. The cleaned isolates were kept on nutrient agar plates and stored at 4 °C, and a copy of each isolate was stored as a glycerol 40% stock at −30 °C.
2.2. Molecular Characterization of Selected PSB
The isolates showing the most pronounced P solubilizing activity (indicated by the solubilization index) were selected for further analysis. DNA extractions from these isolates were set, the extraction of DNA was achieved using the PureLink® Genomic DNA Mini Kit (Invitrogen, Waltham, MA, USA, K1820-01) following the steps defined by the manufacturer, modified for Gram-negative bacteria. PCR amplification of the 16S rRNA of the bacterial strains was made using the DreamTaq PCR Master Mix (Invitrogen), containing of 22 mM Tris-(hydroxymethyl) aminomethane hydrochloride (pH 8.4), 55 mM potassium chloride, 1.65 mM magnesium chloride, 220 μM 2R,3S,5R)-5-(2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-3-hydroxyoxolan-2-yl]methyl, 220 μM 2R,3S,5R)-5-(6-aminopurin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl, 220 μM hydroxy-[[(2R,3S,5R)-3-hydroxy-5-(5-méthyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]méthoxy]phosphoryl, 220 μM 2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]méthoxy-hydroxyphosphoryl, and 22 U recombinant Taq DNA Polymerase/mL.
The universal primers 27F (f
orward) (5′AGAGTTTGAT CCTGGCTCAG-3′) and 1492R (
reverse) (5′-ACGGTTAC CTTGTTACGACTT-3′) were used to amplify a 1500 pb fragment, that corresponds to the genes of the bacterial 16S rRNA. PCR products were purified with the PureLinkTM Quick Gel Extraction & Purification combo kit (Invitrogen K220001), according to the manufacturer’s recommendations. Sequencing was made via primers 27F and 1492R, and carried out according to the Sanger technique adapted by the Big Dye Terminator V3 sequencing kit [
19]. The ABI3730 DNA sequences permitted the automatic analysis of sequence reactions. The crude electropherograms were studied by MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets (Kumar, Stecher, and Tamura 2015), downloaded from
www.megasoftware.net free of charge [
19]. The consensus sequence from the f
orward and
reverse raw sequences were obtained for each strain; then, it was compared to other sequences using the BLAST server (
blast.ncbi.nlm.nih.gov) to determine their phylogenetic affiliation [
19]. The phylogenic tree was built via the neighbor-joining method [
20].
2.3. Morphological, Biochemical Characterization
The selected strains were cultured on nutrient agar to study their morphological characterization, whereas for the observation of cell structure, Gram staining method was used. Biochemical characterization was carried out by the API 20 E system (API System, bioMerieux, Montalieu Vercie, France), following the manufacturer’s instructions. The API 20 E system is composed of 20 microtubes with dehydrated substrates inoculated with a bacterial suspension.
2.4. Plant Growth-Promoting Traits of PSB
The isolated strains were tested for their plant growth-promoting traits; for the Indole-3-Acetic Acid (IAA) production, the method described by Gordon and Weber [
21] was approved; 200 µL of fresh bacterial cultures were inoculated in 30 mL of LB broth containing 0.1%
l-tryptophan and incubated in the dark for 72 h in an incubator shaker at 28 °C and 140 rpm/min. The bacterial cultures were centrifuged at 10,000 rpm for 10 min. Then, 2 mL of supernatant was mixed with Salkowski reagent. After 30 min in dark, the optical density was measured at 530 nm using Ultraviolet and Visible Range Spectrophotometers UV-2005 (Spain). The quantity of IAA produced was determined by the standard graph of pure IAA. The siderophore production was determined on Chrome-Azurol S (CAS) medium following the Universal Chemical Assay according to the method defined by Schwyn and Neilands [
22]. The development of yellow-orange halo around the cell was described as positive for siderophore production, and to detect the cyanide production by the strains selected, the method of Bakker and Schippers [
23] was carried out. Plates observed for change in color of filter paper from yellow to orange to brown were described as positive for cyanide production.
2.5. Qualitative Analyses of Potassium (K) Solubilization
The following medium was used to assess the ability of isolate strains to solubilize potassium: 5 g
d-glucose, 0.005 g magnesium sulphate heptahydrate, 0.1 g iron(III) chloride, 2.0 g calcium carbonate, 2.0 g calcium orthophosphates, 20 g agar, and 3.0 g mica as an insoluble K source per liter. The medium was autoclaved for 20 min to sterilize it. The medium was spiked with 0.25% bromothymol blue dye. The inoculated Petri plates were sealed and incubated for 72 h at 30 °C in an incubator [
24]. Following the incubation period, the bacterial isolates’ ability to solubilize K was assessed qualitatively by looking for clear zones and a change in the color of the bromothymol blue dye from greenish blue to yellow.
2.6. Organic Acid Analysis by GC-MS
The PSB strains were grown in NBRIP broth medium for acid organic determination. Before autoclaving, 50 mL of NBRIP broth medium was adjusted to pH = 7.02 in a 250 mL flask. The medium was inoculated with 200 μL of fresh inoculum (1.8 × 108 CFU/mL) and incubated in shaking conditions at 120 rpm/min at 30 °C for 72 h.
Sample preparation: After centrifuging the sample for 5 to 10 min, 5 mL of supernatant was transferred to a Falcon tube. Considering the pH of the sample, 100 μL of sulfuric acid (1 N) was added. The pH was kept between 2 and 4. The samples were filtered in duplicate through a 0.2 µm filter and then, phenol (0.05 M) was added.
Gas Chromatography coupled Mass Spectrometry (GC-MS) [
25] was used to determine the presence of organic acids (acetic acid, formic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, caproic acid, heptanoic acid) in the samples. The calibration curves of the standards were used to quantify the acids.
2.7. Inoculum Preparation
The purified isolates were grown in the NBRIP medium. Each isolate was inoculated into test tubes containing 5 mL of NBRIP liquid medium and incubated for 24 h at 30 °C. Cells were harvested by centrifugation (Sigma 1-15K, Neustadt an der Weinstrasse, Germany) at 6400 rmp for 8 min, washed with 0.9% sterile saline, and were re-suspended to a 0.5 McFarland nephelometer standard to obtain an inoculum ~1.8 × 10
8 CFU/mL [
26].
2.8. Solubilization Test of the Three Forms of Phosphates
The solubilization test of the three forms of phosphates (Ca
3(PO
4)
2, AlPO
4, and FePO
4) by the selected strains was estimated quantitatively and approved by using Erlenmeyer flasks of 100 mL containing 50 mL of liquid NBRIP medium adjusted to pH = 7.0 ± 0.2 before autoclaving amended with 5.0 g/L of calcium phosphate or aluminum phosphate or iron(III) phosphate as a sole source of P; then, inoculated with 200 μL of each isolate and incubated in shaking condition at 120 rpm/min at 30 °C for 7 days; 2 mL of the cultures were taken every 48 h and centrifuged at 10,000 rpm for 15 min. The content of soluble phosphate was estimated according to Murphy and Riley [
27] using the molybdenum blue colorimetric method by measuring the absorbance at a wavelength of 882 nm with Ultraviolet and Visible Range Spectrophotometers UV-2005 (Spain). All treatments were in triplicate. The pH of the samples was also measured every 48 h with a digital pH meter.
2.9. Statistical Analysis
Data were analyzed using SPSS 20 software, and the results were expressed as the means ± standard deviation of three replicates. Data were examined by ANOVA, and post hoc mean comparison was performed by Duncan’s multiple range test at p ≤ 0.05.
4. Discussions
The objective of our study was to isolate PSB from phosphate solid sludge based on the NBRIP medium with Ca
3(PO
4)
2 as the sole source of phosphorus, then to evaluate their ability to solubilize three forms of inorganic phosphates: Ca
3(PO
4)
2, FePO
4, and AlPO
4. From a hundred isolates, we selected nine strains based on their ability of solubilization. Several authors have focused their studies on the solubilization of Ca
3(PO
4)
2, FePO
4, and AlPO
4 [
28,
29,
30,
31]. The strains assessed in our investigation were able to solubilize Ca
3(PO
4)
2; for FePO
4 and AlPO
4, the solubilization was minimal compared to Ca
3(PO
4)
2, as well, the selected isolates showed high efficiency of solubilization for Ca
3(PO
4)
2, since FePO
4 and AlPO
4 have a more complex structure than Ca
3(PO
4)
2. Previous studies reported that the solubilization of Ca
3(PO
4)
2 was the highest. Similar observations were reported by Reyes et al. [
32] and Banik and Dey [
28], when iron phosphate and hydroxyapatite were used. Correspondingly, it has been revealed by Devi and Thakuria [
31], in their investigation on the PSB predominance in rice rhizospheric soils, that there was only 40.7% of 172 isolates dissolved aluminum phosphate (AlPO
4). The soluble-P concentration ranged between 112.51 μg/mL and 174.33 μg/mL for Ca
3(PO
4)
2, 84.15 μg/mL, 34.85 μg/mL for FePO
4, 68.24 μg/mL, and 17.05 μg/mL for AlPO
4. The isolate BM11 showed the highest potential to dissolve P from Ca
3(PO
4)
2, and BT3S171 from AlPO
4 and BM28 from FePO
4. Additionally, in our study, the maximum solubilization of different phosphate sources was generally obtained after 96 h of incubation. On the 6th day, for some strains in all treatments, a decrease in phosphate solubilization had been observed. This decrease might be due to the diminution of nutrients in the medium [
33].
Phosphate solubilization of the three forms of P followed by a decrease in pH of the medium was observed in the range 7.00–3.2. The decreasing pH of medium has likewise been reported from previous studies [
34,
35,
36]. The production of organic acids by bacteria throughout their metabolic process induces the pH to decrease. Many PSB strains have been observed secreting a variety of organic acids, including acetic, citric, formic, oxalic, and formic acid, amongst many others. Organic acids [
1,
25,
37,
38,
39,
40,
41] convert tricalcium phosphate to mono and dicalcium phosphate, allowing plants to receive phosphorus minerals. According to Nahas [
42] and Anand et al. [
43], organic acids generated by bacteria dissolve insoluble phosphate with a decrease in pH, chelation of cations, and interaction with phosphate on sorption sites in the soil. Thus, organic acids produced by PSB metabolism bound or chelate cations that bound P, therefore P solubility increases [
44,
45,
46]. According to Mahidi [
47] and Elfiati et al. [
48], the molecular structure of organic acids, notably the number of carboxyl and hydroxyl groups, has a significant influence on their ability to chelate metal cations. The type and position of the ligand, in addition to the acid’s strength, determine its efficacy in the dissolving process.
In our investigation, PSB isolates produce organic acids in different quantities and types. The results of all isolated strains showed that acetic and isobutyric acid acids were the two major acids produced by all PSBs tested. PSB’s capacity to dissolve P is influenced by the amount and type of organic acids generated. All PSB isolates produced acetic, formic, and isobutyric acid, whereas eight isolates produced caproic acid, seven isolates produced isovaleric acid, six isolates produced propionic acid, and two isolates produced heptanoic acid. Organic acids have different abilities when it comes to releasing P bonds. Moreover, citric acid dissolved P more effectively than oxalic and malic acids, according to Hocking [
49] and Hou et al. [
50], and organic acids that can form a more stable complex with metal cations will be more successful in releasing aluminum and iron from soil minerals, allowing for more phosphorus to be released. In our study, four genera were determined
Pseudomonas,
Serratia,
Pantoea, and
Enterobacter, PSBs are diversified in nature [
51], according to Biswas et al. [
52], Sulbaran et al. [
53], Bendjelloul et al. [
54], and Liu et al. [
36]; bacteria belonging to the genera
Pseudomonas,
Enterobacter,
Serratia, and
Pantoea are potent PSMs. Moreover,
Pseudomonas genera are among the most efficient solubilizers of inorganic phosphate [
55].
The strains investigated were assessed for PGP characteristics (IAA, siderophores, and HCN) and potassium solubilization. In addition to their ability to solubilize inorganic phosphates, PSB efficiency is due not only to their potential to raise P availability, but also to their capacity to produce growth-regulating agents such as IAA, a growth regulator that aids in cell growth and division, stress resistance, root lengthening, nitrogen fixation stimulation, and biosynthesis of various metabolites [
56]. All PSB isolates generate IAA, with different quantities between isolates. Likewise, in the secretion of siderophores, which plays a very important role in the release of iron [
57], siderophores behave as dissolving agents for iron from minerals or organic compounds under conditions of iron restriction. As well, siderophores can form stable complexes with additional metals that are environmentally damaging [
58]. In our study, all PSB isolates produced siderophores. On the other hand, plants require K as the third most important macronutrient. More than three-quarters of the K in agricultural soils are in the form of insoluble organic and inorganic molecules or complexes, which are inaccessible to plants [
59], such as BSP strains, where the use of K-solubilizing bacteria as a biofertilizer might be an environmentally friendly alternative technique for plant K uptake. BSPs have the potential to be a useful biofertilizer. Phosphate-solubilizing, potassium-solubilizing, and significant growth-promotion effects on plant development have been observed for many BSP species. In the present study, we have clearly demonstrated that the isolated PSB can be a potential plant microbial agent that could be used to promote plant growth even in acidic or calcareous soil.