Isolation, Solubilization of Inorganic Phosphate, and Production of Organic Acids by Individual and Co-inoculated Microorganisms

Abstract Phosphate solubilizing microorganisms (PSMs) mineralize phosphate (PO4³−) to make phosphorous available for plant uptake. This study is designed to isolate the PSMs from the subtropical environmental samples. Total 35 bacterial and 18 fungal isolates were found to be effective P solubilizers depend on their ability of P solubilization that were determined qualitatively and quantitatively using National Botanical Research Institute Phosphorus media. Efficient PSMs that were solubilizing phosphate in maximum amount were identified as Pseudomonas aeruginosa, Serratia marcescens, Pseudomonas mosselii, Bacillus licheniformis, Bacillus subtilis, Bacillus species, Aspergillus flavus, Aspergillus foetidus, Aspergillus niger, Aspergillus tubingensis, and Penicillium chrysogenum. Among them, A. tubingensis was observed as the most efficient phosphate solubilizing strain. For the first time, combined phosphate solubilization activity of A. tubingensis with other efficient phosphate solubilizers was checked and found that A. tubingensis and P. aeruginosa acted synergistically and solubilized phosphate in increased amount. High-performance liquid chromatography results revealed that A. tubingensis produced variety of organic acids including succinic, gluconic, oxalic, and citric acids. Scanning electron microscopy results confirmed the presence of firmly attached large fungal hyphae on tri-calcium phosphate (Ca3(PO4)2). Thus, the results suggest that the use of these PSMs either single or combined as bioinoculants will help to increase the availability of soluble phosphorous.


Introduction
Phosphorus (P) is the second most important macronutrient after nitrogen, which plays a significant part in the plant growth and development (Xiao et al. 2020;Zineb et al. 2020). It is a part of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) which is essential for energy transformation and storage (Billah et al. 2019). It also occupies an important place in plant photosynthesis, glycolysis, respiration, formation of the membrane of cells, and the activities of enzymes (Borgi et al. 2020;Manzoor et al. 2017).
Plants can absorb phosphorus only in soluble form and most part of phosphorus in the soils is present in the insoluble form (Sarikhani et al. 2020). The limited concentration of phosphorus in agricultural soil incited farmers to apply chemical P-fertilizers containing solubilized form of phosphorus (triple super phosphate and di-ammonium phosphate; Borgi et al. 2020). However, A large proportion of soluble chemical fertilizer instantly get immobilized and become unavailable for plant after application (Rodr ıguez and Fraga 1999;Sarikhani et al. 2020).
The high amount of soluble inorganic phosphorus (more than 80%) readily precipitates after addition in soil by making the complex compound with aluminum and iron in acidic soil, monocalcium-bicalcium-tri-calcium in alkaline soil and it also adsorbs on aluminum oxides, iron and clays (Lobo et al. 2019). Only 10% of added phosphorus become available for plant uptake whereas the major remaining quantity of phosphorus remained complex into an insoluble form or washed away and causes destructive eutrophication and various environmental problems including acidification of soil, water and air pollution etc (Borgi et al. 2020;Zhao et al. 2019).
A better alternate is to replace phosphorus-based chemical fertilizer with an organic or bio fertilizer. Biological solubilization of phosphate is an important aspect that should be considered while formulating the biofertilizer for the improvement of agricultural crop production (Borgi et al. 2020).
Several soil phosphate solubilizing microorganisms (PSMs) are reported to play a crucial role in the transformation of insoluble phosphates into soluble phosphorus by the production of phosphatase, phytase, organic acids, chelation, mineralization, and lowering soil pH (Kalayu 2019;Xiao et al. 2020 (Acevedo et al. 2014;Manzoor et al. 2017;Panda et al. 2016). Many soil fungal species are also reported as phosphate solubilizing fungi (PSF) such as Aspergilli and some species of Penicillium (Acevedo et al. 2014;Bhattacharjya et al. 2019;Saber et al. 2009).
The potential advantage of phosphate solubilizers in plant productivity and prevention of environment pollution suggest that the research on phosphate solubilizing microorganisms needs to be accelerated greatly (Fatima et al. 2018). It is essential to isolate microorganisms from relevant ecological niches to maximize their chances for use as a means of improved soluble phosphorus ability in the field (Jiang et al. 2020).
Present study was aimed to isolate and characterize the PSMs from subtropical environmental samples including soil, water and fruit-based compost. For the first time, to our knowledge, fruit-based compost especially from subtropical environment was assessed for isolation of PSMs. The study also characterized the individual and co-inoculated PSMs for Phosphate solubilization under in vitro conditions in qualitative and quantitative assays while the ability to produced organic acids were also assessed.

Sample collection and preparation
Garden soil and tap water were collected from the Department of Microbiology, University of Karachi (24 56 0 24 00 N 67 7 0 6 00 E) whereas previously produced fruitbased compost (Danish et al. 2021) samples were also collected in a sterilized glass bottle. Samples were 10-fold serially diluted up to 10 8 times dilution in sterilized distilled water for the isolation of phosphate solubilizing microorganisms (PSMs).

Screening and isolation of PSMs
Phosphate solubilizing bacteria (PSB) and phosphate solubilizing fungi (PSF) were screened through plating method at 30 C for one week on National Botanical Research Institute Phosphorus (NBRIP) agar media consist of Glucose 10 g/l, Ca 3 (PO 4 ) 2 5 g/l, NaCl 0.2 g/l, MgSO 4 7H 2 O 0.25 g/l, KCL 0.2 g/l, (NH 4 )SO 4 0.1 g/l, and Agar 15 g/l (Nautiyal 1999). Mesophillic bacterial and fungal colonies, producing zone of clearance were selected and transferred onto nutrient agar (NA) and Sabouraud's dextrose agar (SDA) media for purification purposes and incubated at 30 C for 24 h and 1 week, respectively. Culture slants were stored at 4 C for further use.

Estimation of phosphate solubilization in solid medium
For estimation of phosphate solubilization index (SI) and efficiency (SE) in solid media, a pinpoint microbial colony was inoculated on NBRIP agar plates and incubation was done at 30 C for 7 and 10 days for bacteria and fungi, respectively. The increase in the colony size and the zone of clearance formation (diameter of the zone of clearancediameter of the colony) were recorded after every two days in millimeter (mm). Based on the diameter of zone of clearance and the diameter of the colonies, SI and SE were estimated by using the following formula (El-Hamshary et al. 2019;Panhwar et al. 2012

Estimation of phosphate solubilization in liquid medium
The single bacterial colony and fungal spores (10 5 ml À1 ) was inoculated in 100 ml NBRIP broth (pH 7.0) amended with tri-calcium phosphate in triplicates. The fungal spores were confirmed by spore count using a hematocytometer (Smith et al. 1988). Flasks were placed at 30 C in a shaking incubator at 150 rpm for 12 days. The amount of the phosphate released was estimated on the 3rd, 6th, 9th, and 12th day. Each day 1 ml sample was taken and centrifuged at 10,000 rpm for 10 min at room temperature then the supernatant was collected and filtered through 0.45 mm what-manV R filter paper to remove cells and insoluble contents. pH was also regularly noted using the digital pH meter. The supernatants were used to estimate released phosphate spectrophotometrically at 880 nm according to the standard molybdenum blue method of Murphy and Riley (1958). The working standards of 0.2, 0.4, 0.6, 0.8, and 1.00 PPM were prepared by making a 100 ppm stock solution of KH 2 PO 4 , and the solubilization of PO 4 was quantified by matching the absorption of standards with the absorption of samples (Mujahid et al. 2014;Zineb et al. 2020).

Evaluation of organic acids
Production of organic acids by phosphate solubilizing microorganisms were measured through high-performance liquid chromatography (HPLC) using method described previously by Jiang et al. (2018) with slight modifications. Crude supernatant filtrates (collected from 'Estimation of phosphate solubilization in liquid medium' section) containing the secreted PSMs organic acids were centrifuged for 15 min at 10,000 rpm and then supernatants were filtered in duplicate via 0.22-mm whatman V R filter paper. HPLC Analyst equipped with a UV visible detector and auto sampler by Perkin Elmer was used with software Total Chrome work station version 6.3.1. A column with 4.6 Â 250 mm dimension, Spheri-5 ODS reverse phase C18, with particle size 5 micron was operated using a mobile phase mixture of 80 mM phosphoric acid (H 3 PO 4 ) pH 2.0 and acetonitrile (C 2 H 3 N) in a ratio 88:22 v/v. The purified culture filtrate and standard solution samples were proceeded for 10 min with a 1 ml/min flow rate and 10 ml of an injection volume. Analytical grade standards (Sigma) of the following organic acids were used: succinic, gluconic, oxalic, and citric acids. Different unknown organic acid concentrations in culture filtrate were analyzed by matching the retention time of the eluted peaks corresponding with the standard organic acids peaks at k 210 nm (Bakri 2019;Kumari et al. 2008;Mardad et al. 2013).

Estimation of combined phosphate solubilization activity of PSMs
Combined phosphate solubilization activity of isolated phosphate solubilizing bacteria and phosphate solubilizing fungi were estimated to check the maximum phosphate solubilizing activity of bacteria and fungi Saxena et al. 2016). Single bacterial colony of AAB12 and AAC1 whereas 10 5 ml À1 fungal spores of AAP8 and AAP11 were used for inoculation. Culture of AAP11 and AAC1, AAP11 and AAB12, AAP11 and AAP8, and AAP11, AAB12, AAP8 and AAC1 were inoculated in 50 ml of sterile NBRIP liquid broth medium amended with tri-calcium phosphate in triplicates. Un-inoculated sterilized medium was set out as control. Flasks were placed at 30 C with constant shaking at 150 rpm for 12 days. After incubation, samples were centrifuged at 10,000 rpm for 10 min and clear supernatants were used to detect pH and released phosphorous in the medium (as mentioned in 'Estimation of phosphate solubilization in liquid medium' section).

Scanning electron microscopic (SEM) morphology
SEM was performed to evaluate the interaction of PSMs with Phosphates using method described previously by Islama et al. (2007). Microbial biomass (collected from 'Estimation of phosphate solubilization in liquid medium' section) and lyophilized. Cells fractions were then placed on a gold coated brass stub under vacuum, and observed under scanning electron microscope at different resolutions (JEOL SEM6380a, Japan).

Identification of the PSMs
Extraction of DNA was done according to manufacturer's instructions. Phosphate solubilizing bacterial DNA was extracted by using the DNA Purification Kit of Wizard V R Genomic Promega (Cat no A1120) and phosphate solubilizing fungal DNA was extracted by using, Ez-10 spin column fungal genomic DNA mini-prep extraction kit (Bio Basic). For the identification of phosphate solubilizing bacteria, 16S rDNA gene was amplified by using universal primers 27 F (5 0 -AGAGTTTGATCMTGGCTCAG-3 0 ) and 1492 R (5 0 -TACGGYTACCTTGTTACGACTT À3 0 ) (Frank et al. 2008) whereas internal transcribed spacer (ITS) regions were amplified for the identification of phosphate solubilizing fungi using ITS1 forward (5 0 -TCCGTAGGTGAAC CTGCGG-3 0 ) and ITS4 reverse (5 0 -TCCTCCGCTTATTG ATATGC-3 0 ) primers (Danish et al. 2021;White et al. 1990). The PCR mixture (50 ml) contained $10 ng DNA, 1 mM forward and reverse primer each, 25 ml Go Taq PCR Master Mix (Penicon) and top up to 50 ml by nuclease free water. For the amplification of bacterial 16S rDNA, initial denaturation was done at 95 C for 2 min, the repetitive 35 cycles include denaturation at 95 C for 15 sec annealing at 55 C for 30 sec, extension at 72 C for 2 min. Final extension was performed at 72 C for 6 min (Fredriksson et al. 2013). For the amplification of fungal ITS region, initial denaturation was done at 95 C for 5 min. The reaction proceeded for 35 cycles. Denaturation at 95 C for 30 sec, annealing at 55 C for 60 sec, elongation at 72 C for 120 sec and final extension at 72 C for 7 min (Danish et al. 2021). For sequencing, amplified PCR products were sent to the Beijing Genomics Institute (BGI), China. The Bio-Edit Sequence Alignment Editor (7.2.5, Ibis Biosciences. USA) was used to analyze the sequenced data. The obtained nucleotide sequences were aligned against the available sequences in the database that were checked by using BLAST (Basic Alignment Search Tool) from the NCBI (National Center for Biotechnology Information) server (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were submitted for acquisition of accession numbers at NCBI.

Statistical analysis
All the experiments were run in triplicates. Mean and standard deviation (x þ SD) were calculated using Microsoft Excel (2010).

Screening and isolation of PSMs
The phosphate solubilizing microorganisms (PSMs) were screened and a total of 134 bacterial and 49 fungal colonies with different morphologies were obtained on NBRIP media at 30 C. Out of total recovered colonies, 35 bacterial (AAB1-AAB28, AAC1-AAC4, AAW1-AAW3) and 18 fungal (AAP1-AAP18) colonies showed vivid zone of clearance. These colonies were isolated and selected as phosphate solubilizing microbial strains (Table 1).

Phosphate solubilization in liquid medium
Isolated PSMs were further subjected for analysis for solubilization of phosphate in NBRIP broth medium consisting Ca 3 PO 4 as a source of insoluble mineral phosphate to quantify the amount of released soluble phosphate in a medium. Results of the phosphate solubilization at different intervals are presented in Table S1. In this study, AAB4, AAB5, AAB6, AAB8, AAB9 AAB12, AAB13, AAB14, AAC1, and AAW1 bacterial strains whereas AAP3, AAP4, AAP7, AAP8, AAP11, and AAP13 fungal strains exhibited highest phosphate solubilization activity in liquid broth medium (Table 1). No solubilization of P occurred in controls. Among phosphate solubilizing bacterial isolates, the maximum P solubilization occurred by Bacillus species AAB12 (1.94 mg/ l). Among phosphate solubilizing fungal isolates, the maximum P solubilization occurred by Aspergillus species AAP3 and AAP4 (1.61 and 1.21 mg/l) followed by AAP7 and AAP11 which solubilized maximum phosphate (1.05 and 1.02 mg/l). Drop in pH of liquid NBRIP broth medium from 7 was also recorded in the range between 3.0 and 6.6 during phosphate solubilization.

Identification of PSMs
The most potent ten PSB and six PSF strains were selected for molecular identification ( Table 2). The most recovered genera of phosphate solubilizing bacteria belong to the Bacillus and Pseudomonas. In fungi, the most recovered genera were Aspergillus and Penicillium. The phylogenetic analysis of the isolates shows 96-100% sequence homology with the reference sequences from the NCBI data base (Figures 3  and 4 and Table S2).

Organic acids detection by HPLC
The secretions of organic acids by isolated PSM strains were examined in NBRIP broth medium. HPLC determination unfolded the existence of different organic acids that confirm the role of insoluble tri-calcium phosphate solubilization of isolated strains. Concentrations of organic acids were checked after 12 days of incubation. Presence of seven different organic acids was observed, among them four were    oxalic, succinic, gluconic and citric acid and three were unknown organic acids (unknown 1, unknown 2, and unknown 3). Retention time of oxalic, gluconic, citric, and succinic acid was 2.07, 2.19, 2.47, and 2.91, respectively, whereas retention time of three unknown organic acids were 3.1, 4.1, and 7.3 for unknown 1, unknown 2, and unknown 3, respectively. Different strains showed the presence of multiple organic acids (Table 3) ( Figure S1). Fungal strain Aspergillus tubingensis AAP11 produced a maximum number of organic acids with a highest pH reduction (from 7 to 3.2) along with an excessive level of P solubilization. Isolated strains showed the presence of succinic acid in maximum amount (3.42-7.9 mg/ml). In this study, largest amount of succinic acids (6.71-7.9 mg/ml) was produced by Aspergillus flavus AAP3 and Penicillium chrysogenum AAP8 among all the isolates. After succinic, gluconic, oxalic, and citric acids, were produced. Gluconic acid was produced by Aspergillus foetidus AAP4, A. tubingensis AAP11, and Aspergillus niger AAP13. Among all the isolates, citric acid was produced only by A. flavus AAP3 and A. tubingensis AAP11.

Combined phosphate solubilization activities of PSMs
Combined phosphate solubilization activities was checked to analyze the symbiotic relationship among PSMs so it could also be used as bioinoculants. In this study, A. tubingensis AAP11 reported as most efficient phosphate solubilizing strains both qualitatively and quantitatively therefore for the first time, combined phosphate solubilization activities of A. tubingensis AAP11 was checked with Pseudomonas aeruginosa AAC1, Bacillus sp. AAB12, and P. chrysogenum AAP8. Co-inoculation of A. tubingensis AAP11 and P. aeruginosa AAC1 showed the best P solubilization (2.38 mg/l ± 0.56) with a pH reduction 3.15 ± 0.08 from initial 7 suggesting a synergism among the tested organisms as compared with other tests (Table 4). The lowest phosphate solubilizing activities were observed in NBRIP broths inoculated with three efficient strains i.e., 1.83 mg/l ± 0.31 with a pH reduction 5.19 ± 0.18. Synergism was also seen when A. tubingensis AAP11 inoculated with Bacillus sp. AAB12 and Penicillium sp. AAP8 but maximum solubilization of phosphate was obtained via co-inoculation of A. tubingensis AAP11 and P. aeruginosa AAC1 that acted synergistically with each other and solubilized phosphate in maximum amount.

PSF colonization on tri-calcium phosphate surfaces with SEM
The colonization of PSF was examined under a scanning electron microscope. The existence of firmly attached large fungal hyphae (Figure 5(a-f)) on tri-calcium phosphate was observed under SEM in contrast with un-inoculated control ( Figure 5(g)) where no such fungal cells were observed. The micrographs illustrate the fungi grew with the aggregate emergence and intact morphological features in NBRIP medium on Ca 3 PO 4 .

Discussion
The screening of the phosphate solubilizing microorganisms (PSMs) was done on NBRIP medium as Nautiyal (1999) developed an efficient media for the screening of PSMs. El-Hamshary et al. (2019) reported that zones of clearance are the indications of dissolving phosphate elements present in the medium (Figures 1 and 2). This phosphate solubilization estimation method has been used by many researchers (     A. niger AAP13 found to be recorded 2.4. Astriani et al. (2020) suggested that S.I may vary because of the production of organic acids. LA et al. (2018) reported SI for different Aspergillus species isolated from different soil samples ranged from 2.1 to 2.4, these observations are in consistent with the present study. Rodr ıguez and Fraga (1999) reported that identification of PSMs should also be confirmed through phosphate solubilization in liquid medium since contradictory results can be obtained through plate screening.
Among phosphate solubilizing bacterial isolates, the maximum P solubilization occurred by Bacillus species. Among phosphate solubilizing fungal isolates, the maximum P solubilization occurred by Aspergillus species. The results are in comparison with previously published data (Afzal et al. 2013;Babu et al. 2017;Boroumand et al. 2020;Teng et al. 2019). Chiadikobi et al. (2014) recorded maximum solubilization of tri-calcium phosphate inoculated with PSF in liquid NBRIP broth medium on 12th day. Higher solubilization was observed by Aspergillus species, these finding are in consent with Achal et al. (2007) who reported that the Aspergillus sp. showed the highest available phosphate when tri-calcium phosphate was used. Das et al. (2013) reported A. niger as the most efficient phosphate solubilizing strain among the tested fungi. Astriani et al. (2020) suggested that the concentration of dissolved phosphate rely on sources of phosphate, microorganism's growth and condition of culture. Alori et al. (2017) demonstrated that soil fungi perform an important role in inorganic P solubilization as they produce more organic acids than bacteria because fungi have ability to traverse long distances in the soil as compared to bacteria. Consequently, PSF considered as primary candidate in the group of PSM (Li et al. 2016).
Drop in pH of liquid NBRIP broth medium was also recorded during phosphate solubilization. Obtained results are in consistent with several researchers (Gupta et al. 2007;LA et al. 2018;Saber et al. 2009) who reported a remarkable reduction in the pH while, pH stand the constant in the uninoculated (control) during incubation days. Several reports suggests that production of organic acids and acidification of medium facilitates the solubilization of P from its compound (Behera et al. 2016;Boroumand et al. 2020;Paul and Sinha 2017). Reyes et al. (1999) also reported the production of organic acids reduces the pH of liquid medium consisting insoluble phosphates. Acidification can also occur by ammonium assimilation in microbial cells followed by the release of the protons that solubilize the phosphorous (Alori et al. 2017) as well as by chelation of the cations bound of phosphate (Kalayu 2019). Illmer and Schinner (1995) reported the release of protons leading respiration or NH 4 þ assimilation may be one of the cause of lowering the pH. Li et al. (2016), suggested that the pH value in PSF culture medium could be drop to 1 due to 10Â higher production of organic acids in contrast with bacteria.
The most recovered genera of PSB belong to the Bacillus and Pseudomonas as it has frequently been reported as phosphate solubilizing bacteria (Baliah and Begum 2015;Panhwar et al. 2012). Pseudomonas and Bacillus are the most ubiquitously present genera in water and soil (Billah et al. 2019). In fungi, the most recovered genera were Aspergillus and Penicillium. These are the most commonly recovered fungal from compost. Their extensive sporulation property favors their growth on SDA Zafar et al. 2014). These outcomes are in agreement with several researchers (Chuang et al. 2007;LA et al. 2018;Onyia et al. 2015), who also reported Aspergillus and Penicillium as the most important PSF genera isolated from the different environmental localities and habitats and identified as the most potent isolates.
It has been well known that the microorganisms solubilize tri-calcium phosphate through the acidification of medium via different organic acids productions (Rodr ıguez and Fraga 1999). Organic acids can solubilize phosphate either as an outcome of anion exchange or chelation of Al, Ca, or Fe, ions that linked with the insoluble P (Mardad et al. 2013). Fungal strain A. tubingensis AAP11 produced a maximum number of organic acids. The secretions of multiple organic acids with a pH reduction in a culture medium as mentioned earlier thereby solubilizing the insoluble tricalcium phosphate (Chen 2006). The findings of this analysis are also in agreement with the observations of several researchers (Banik and Dey 1982;Parks et al. 1990;Rashid et al. 2004;Singal et al. 1994;Whitelaw et al. 1999), who reported the production of organic acids such as succinic, oxalic, gluconic, and citric acids etc by phosphate solubilizing microorganisms. Largest amount of succinic acids was produced by A. flavus AAP3 and P. chrysogenum AAP8 among all the isolates. Bakri (2019) also reported maximum amount of succinic acid secreted by Aspergillus sp., when TCP was used. After succinic, gluconic, oxalic, and citric acids, were produced. Gluconic acid was produced by A. foetidus AAP4, A. tubingensis, AAP11, and A. niger AAP13. Goldstein (1995) suggested that gluconic acid production is the outcome of the activities of periplasmic or cell membrane bound NADP-dependent glucose dehydrogenase (GDH) activities though that PSMs liberates P from insoluble mineral phosphate. Glucose is transformed into gluconic acid that creates a transmembrane proton that can be used for membrane bio-energetic and transport functions and the gluconic acid protons are free for solubilizing phosphate (Mardad et al. 2013). Alam et al. (2002) observed oxalic acids to be the most abundant organic acids secreted by the microorganisms that are present in the rhizosphere of maize. Among all the isolates, citric acid was produced only by A. flavus AAP3 and A. tubingensis AAP11. Dorcas et al. (2020) also reported citric acid in very low concentration and even not detected by some strains and suggested that citric acid is originated from Krebs cycle, therefore, its synthesis, requires very high energy for the cell so cell abstains from its release until necessarily required. The secretions of organic acids supports the results of acidification or pH reduction in medium (Park et al. 2016). Production of different organic acids demonstrated evidence that isolated PSM strains have effect on phosphate solubilization. In this study, HPLC results revealed that isolated PSMs produced succinic and gluconic acids in relatively high concentrations than oxalic and citric acids. Results also revealed that A. tubingensis AAP11 produced a maximum number of organic acids and showed the potential for acid secretion.
Previously, our work (Ahmad et al. 2022) suggests that generally regarded as safe (GRAS) microorganisms can be used as biofertilizers so venturing the organic acid production from novel isolates would help us in designing better biofertilizer.
The interactions of microorganisms are important in an ecosystem particularly in soil. In soil, bacteria and fungi share a habitat known as bacterial and fungal interface in which bacteria usually present on fungal hyphae or spores in association with mycorrhizal roots where these microbes work in the soil in different ways such as symbiosis, saprotrophy, and pathogenicity (Hayat et al. 2017). In a symbiotic relationship of PSM with plants, microbes provide soluble P whereas plants provide carbon (usually sugars) for their metabolism and growth (Becquer et al. 2014). Co-inoculation of A. tubingensis and P. aeruginosa showed the best P solubilization. Saxena et al. (2016) found Pseudomonads and Aspergilli acted synergistically with each other and best P solubilizers when co-inoculated. Lee et al. (2016) clearly depicted that co-inoculation enhances P solubilization in a liquid culture medium. Lowest phosphate solubilizing activities were observed in broths inoculated with three efficient strains. These results are in agreement with Mohamed et al. (2019) who reported no significant increase in the soluble phosphorus in culture medium due to the consumption of phosphorous or the depletion of nutrients such as carbon source that is required for the production of organic acids. Present study, suggest that the maximum solubilization of phosphate can be obtained via co-inoculation of A. tubingensis and P. aeruginosa that acted synergistically with each other and solubilized phosphate in maximum amount.
Adhesion of fungal culture and Ca 3 PO 4 crystals in medium indicates that the fungal cultures represent an intimate association with the crystals of phosphate required for the degradation of phosphates, which also indicates the increased concentration of soluble P in Ca 3 PO 4 amended medium (Jayashree et al. 2011).

Conclusion
It is concluded from the present study that Bacillus sp. (AAB12), P. aeruginosa (AAC1), P. mosselii (AAW1), A. flavus (AAP3), A. foetidus (AAP4), A. niger (AAP13), and A. tubingensis (AAP7 and AAP11) are the best P solubilizers for increasing the bioavailability of phosphorus. Among them, A. tubingensis was reported as the most efficient phosphate solubilizing strain. To the best of our knowledge for the first time organic acid production by A. tubingensis and its combination with other phosphate solubilizing bacterial isolates including P. chrysogenum, P. aeruginosa and Bacillus sp. was checked and found that A. tubingensis and P. aeruginosa acted synergistically with each other. HPLC results also revealed that A. tubingensis produced a maximum number of organic acids and showed the potential for acid secretion that would help us in designing better biofertilizer to increase the availability of soluble phosphorous. Since the isolates have environmental origin, they will not pose any threat to the environment. Further screening of PSM strains is required so that can also be utilized as potential phosphate solubilizers in the development of phosphatic biofertilizer to fulfill the demand for Phosphorus in the improvement and sustain ability of soil fertility.