Zinc phosphate dissolution by bacteria isolated from an oligotrophic karst cave in central China

Abstract

Biogeochemical processes are fundamental to sustain the ecosystem in subsurface caves, but to date they are still far from well understood. To investigate microbially mediated phosphorus and zinc cycles, we isolated three bacterial strains from the dripping water in Heshang cave, central China, identified as Exiguobacterium aurantiacum E11, Pseudomonas fluorescens P35, and Pseudomonas poae P41, respectively. Microbial capabilities in the dissolution of phosphorus-containing minerals were tested with zinc phosphate (Zn₃(PO₄)₂) in batch culture at 30°C. A spectrophotometer, atomic absorption spectrum, and scanning electronic microscopy were used to measure the microbial growth, soluble Zn(II) concentration, and to observe the morphology of Zn₃(PO₄)₂ before and after microbial dissolution. P. fluorescens and P. poae, the well-known phosphorus solubilizing bacteria (PSB), are observed to solubilize Zn₃(PO₄)₂ with an efficiency of 16.7% and 17.6%, respectively. To our knowledge, E. aurantiacum is firstly reported in this study to dissolve phosphorous-containing minerals with a higher efficiency of 39.7%, expanding our understanding about the ubiquitous occurrence of PSB in natural environments. Aqueous Zn(II) concentration positively correlates with H⁺ activity, confirming the presence of acidification mechanisms widely exploited by PSB. Few itching pits were observed on the surface of Zn₃(PO₄)₂ after microbial dissolution, inferring that microbial dissolution is not always associated with the direct contact with minerals. Even though the soluble Zn(II) concentration reached up to 370 mg/L in the system inoculated with E. aurantiacum E11, inhibition of microbial growth was not detected by spectrophotometer. Our laboratory data revealed the importance of microbially-mediated P and Zn cycles in the subsurface ecosystem.

Keywords

karst cave / phosphate solubilizing bacteria (PSB) / zinc toxicity / biogeochemical process / subsurface biosphere

Introduction

Karst areas account for 21% terrestrial land surface area (Engel, 2010). Subsurface karst caves are usually nutrient-limited due to the lack of production from photosynthesis (Lee et al., 2012), though some nutrients might be transported into the caves by underground rivers, by dripping water from overlying soils, or by wind from the outside (Chelius et al., 2009). In particular, due to the low solubility of phosphorous (P)-containing minerals in alkaline conditions, phosphorous might be the key limiting factor for microbial growth in karst areas. Phosphorous in soils is also a limiting factor for plant growth particularly under alkaline conditions (Feng et al., 2004), which leads to the annual application of nearly 30 million tons of phosphorous fertilizer to enhance plant productivity in the world. However, as much as 80% of phosphorous fertilizer becomes immobile and unavailable due to adsorption, precipitation, or conversion to organic forms (López-Bucio et al., 2000), and phosphorus solubilizing bacteria (PSB) are proposed to play a major role in the solubilization of soil unavailable phosphorus (Cunninghum and Kuiack, 1992; Kang et al., 2002; Mikonova and Novakova, 2002; Pradhan and Sukla, 2005; Khan et al., 2007). Nevertheless, less is known whether microbes also play significant roles in phosphorus release in alkaline karst caves.

Even though the phosphorous release from P-containing minerals has been studied extensively (Peix et al., 2001; Welch et al., 2002; Pérez et al., 2007; Rosling et al., 2007; Tao et al., 2008; Singh et al., 2011; Xiang et al., 2011), the behavior of the associated cations released from P-containing minerals, which might also greatly affect the growth of plant or microbes, was not well addressed (Fasim et al., 2002; Saravanan et al., 2007). For example, Zn released from zinc phosphate in aquatic environments and soils can affect the productivity and biodiversity of these ecosystems due to its dual characteristics; zinc is required by many enzymes in trace amounts (Hughes and Poole, 1989), but is toxic in many instances even in relatively low concentrations (McGrath et al., 1995; Atmaca et al., 1998; Bong et al., 2010).

As the important bioavailable phosphorous producers, PSB were hypothesized to exist in dripping water because they are required to sustain karst cave ecosystems. The associated cations released due to the activity of PSB will also affect the microbial activity when the cation concentration reaches a certain level. We thus isolated bacteria from the dripping water in Heshang cave, Hubei, China to investigate their capability to decompose P-containing minerals and to evaluate the cation effect on microbial growth. The results will provide insights into the ongoing biogeochemical process in subsurface environments and help us to understand the maintaining of the ecosystems.

Materials and methods

Sampling

The bacterial strains were isolated from the dripping water of the Heshang cave, which is situated in central China (30°27′N, 110°25′E; 294 m) in the middle reaches of the Yangtze Valley, a region strongly impacted by the East Asian Monsoon (Hu et al., 2008). Dripping water was collected into a 10 L sterilized plastic cask by an aseptic funnel in March 2010 and taken back to the geomicrobiology laboratory at the China University of Geosciences (Wuhan) at 4°C in a cooler. Isolation of bacteria from the water sample was conducted as soon as possible.

Culture media

An oligotrophic medium (Liu et al., 2010) was used for the isolation of bacteria from dripping water in Heshang cave, which contains nystautin (0.006 g/L), pyruvate (0.05 g/L), and agar (15 g/L). The filtered dripping water from Heshang cave was added to this medium.

R₂A medium (Bidle, 2007) was used for the purification of bacterial isolates from oligotrophic conditions. It contains (per liter) 0.5 g yeast extract, 0.5 g peptone, 0.5 g tyrosine, 0.5 g glucose, 0.5 g soluble starch, 0.5 g sodium pyruvate, 0.3 g K₂HPO₄, and 0.05 g MgSO₄. The BTG medium employed for the PSB screening contains (per liter) 10 g peptone, 10 g D-glucose, and 15 g agar. When required, a proper amount of finely ground zinc phosphate was added with thorough stirring to obtain a homogeneous suspension with a final concentration of 5 mmol/L. Experiments in liquid BTG medium amended with Zn₃(PO₄)₂ were also performed to screen the PSB. The dissolution of zinc phosphate was performed in a defined mineral salts medium (MSM), with glucose as the sole carbon source and 5 mmol/L suspended zinc phosphate was added as well. The MSM medium contains (per liter): glucose 10 g, (NH₄)₂SO₄, 5 g, KH₂PO₄ 2 g, K₂HPO₄ 2 g, MgSO₄·7H₂O 250 mg, FeSO₄·7H₂O 10 mg, MnSO₄·7H₂O 10 mg, CaCl₂·2H₂O 10 mg, ZnSO₄·7H₂O 30 mg, H₃BO₃ 100 mg, CoCl₂·6H₂O 117 mg, CuCl₂·2H₂O 30 mg, NiCl₂·6H₂O 10 mg, and NaMoO₄·2H₂O 100 mg (Di Simineet al., 1998).

Isolation and identification of bacteria from dripping water

A 10 mL water sample was filtered through 0.22 μm cellulose acetate membrane, followed by immediate attachment to the oligotrophic medium. After three weeks’ incubation at 25°C, genome DNA was extracted from visible individual colonies in different plates for 16S rDNA sequencing. The 16S rDNA gene was amplified with 27F (5′- AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′- GGCTACCTTGTTACGACTT-3′) primers. The reaction conditions were as follows: 95°C for 3 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 48°C for 1 min and primer extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min. The gene sequencing was performed at Nanjing Genscript Company with the ABI3730 machine. Taxonomic analysis was conducted with RDP 10<FootNote>

http://rdp.cme.msu.edu/

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http://www.ncbi.nlm.nih.gov/

</FootNote> with 97% similarities. Phylogenetic analyses were conducted using MEGA version 5.0<FootNote>

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</FootNote>. The phylogenetic tree was constructed by the neighbor-joining method using the distance matrix from the alignment. Distances were calculated using the Kimura method.

Screening of phosphate-solubilizing bacterial strains

Strains from R₂A medium were streaked on the BGT plates and transferred into BGT liquid medium, both of which were amended with zinc phosphate to screen PSB. The plates were incubated at 30°C for 72 h. The liquid was incubated at 180 r/min and 30°C. Visible transparent dissolution halos on BGT plates will form, and aqueous Zn(II) concentration will increase in the liquid culture if the strains are able to solubilize zinc phosphate.

Zinc phosphate dissolution by different PSB strains

Zinc phosphate tetrahydrate powder was commercially purchased (Sinopharm Chemical Reagent Co. Ltd) and subjected to the dissolution experiment conducted in a MSM medium in a batch culture. The inoculum (2.5%) was transferred from R₂A overnight culture to 400 mL of liquid MSM medium supplemented with 5 mmol/L zinc phosphate. The cultures were incubated in an orbital incubator at 150 r/min and 30°C for approximately 12 days. Samples were collected at 12 h intervals for the measurement of pH, optical density (600 nm), and Zn content. All experiments were run in duplicate, and data presented here are the average of the duplicates. OD600 of the cultures were used as parameters for microbial growth. Zn content in the solution was indicative of the dissolution of zinc phosphate.

Analytical methods

An aliquot of 10 mL culture was collected periodically for pH measurement with a pH meter (Delta 320, Mettler-Toledo, Columbus, OH). One milliliter of sample was used for OD measurement with a spectrophotometer (TU-1800, Beijing pgeneral Company Limited). About 10 mL of the cultures were centrifuged for 20 min at 5000 r/min, and the supernatant was used for aqueous Zn analysis with Atomic Absorption Spectrometry (AAS, Hitachi 180-70, Japan). Samples of zinc phosphate residue were mounted on Al-stubs with double-sided carbon tabs and coated with a thin layer of gold. The morphology images were collected by Scanning Electronic Microscope (SEM, JSM-35CF with 15-39 mm of working distance, 0-39 KV and 6 nm of resolution, Japan).

The AAS and SEM analyses were conducted at the State Key Laboratory of Geological Processes and Mineral Resources in China University of Geosciences (Wuhan). Other analyses were conducted in the geomicrobiology laboratory in the State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences (Wuhan).

Results

Strain screening and identification

Isolated strains of E11, P35 and P41 showed potential ability to dissolve Zn₃(PO₄)₂ by forming transparent halos on BGT plates and increasing the concentration of aqueous Zn in BGT liquid medium. The 16S rDNA gene sequences of the three strains showed 99%, 100%, and 98% identity with the typical strains of Exiguobacterium aurantiacum (M-4), Pseudomonas fluorescens Pf-5 and Pseudomonas poae NBB19, respectively (Fig. 1). The three strains were used for further investigation on P-containing mineral dissolution experiments.

Microbial growth and pH variation

P. poae P41 started to grow after 55 h’ incubation, and then increased exponentially in MSM amended with Zn₃(PO₄)_2. It reached the plateau after 126 h (Fig. 2A). E. aurantiacum E11, P. fluorescens P35 grew slowly for the first 137 h and 156 h, respectively. The growth leveled off after 211 h for E. aurantiacum E11 (Fig. 2A). The growth of P. fluorescens P35 reached a plateau at 201 h and lasted 25 h. After that a re-growth was observed in P. fluorescens P35 and continued to the end of the experiment (Fig. 2A).

In the abiotic system, pH remained quite stable around 6.0 (Fig. 2B). The pH values in microbial inoculated systems were close to those in the abiotic control at the initial microbial growth, but decreased sharply within the exponential phase, with the final pH being 3.87, 4.01 and 4.51 in E. aurantiacum E11, P. fluorescens P35 and P. poae P41 inoculated systems, respectively (Fig. 2B).

Aqueous Zn(II) concentration

The initial Zn(II) content was around 1 mg/L for the medium (Fig. 2C). Variation of Zn(II) concentration in microbial inoculated systems shows good coupling with the microbial growth and pH (Fig. 2). In P. poae P41 inoculated system, the Zn(II) concentration was observed to keep unchanged before 42 h when pH was around 6.0, followed by a slow increase to 89.0 mg/L (90 h) and a sharp increase to 164 mg/L (126 h, Fig. 2C) which is consistent with the pH decrease in the system (Fig. 2B). The Zn(II) content leveled off after 126 h when the pH values stabilized in the system.

The aqueous Zn(II) content in the other two inoculated systems shows that the variation trends were comparable with that in P. poae P41 inoculated system. However, the onset time of Zn(II) increase and the final concentration of aqueous Zn(II) were different. In E. aurantiacum E11 inoculated systems, the Zn(II) concentration started to increase at 108 h with the value of 370 mg/L at the final stage (Fig. 2C). In contrast, the Zn(II) concentration in P. fluorescens P35 inoculated systems was found to increase at 157 h and maintain 163.5 mg/L at the end (Fig. 2C).

The aqueous Zn(II) concentration was found to show a positive correlation with the H⁺ activity calculated from pH values measured, and the correlative coefficient R² are 0.848, 0.842 and 0.990 for E. aurantiacum E11, P. fluorescens P35, and P. poae P41, respectively (Fig. 3). In comparison, a stronger positive correlation (R² = 0.994) was observed in the abiotic control when HCl was used to acidify the solution to release Zn(II) from zinc phosphate (Fig. 3).

Morphology of zinc phosphate

Particles of Zn₃(PO₄)₃ collected at the end of abiotic and biotic experiments were subjected to SEM observation. Zinc phosphate particles show a large size with smooth surfaces in the abiotic system (Fig. 4A). Bacteria were found to congregate (Fig. 4B) or attach on the surface of the zinc phosphate particles (Fig. 4C). In the abiotic system, particles are characterized by relatively sharp edges, clear and neat surfaces. In the biotic systems, the particle edges became obtuse, and some itching pitches with sizes close to that of bacteria were observed on the surface of some particles (Fig. 4C).

Discussion

Wide occurrence of PSB strains

Previously, many bacterial genera were demonstrated to be able to solubilize unavailable phosphate in soils, including Bacillus, Pseudomonas, Erwinia, Agrobacterium, Serratia, Flavobacterium, Enterobacter, Micrococcus, Azotobacter, Bradyrhizobium, Salmonella, Alcaligenes, Chromobacterium, Arthrobacter, Streptomyces, Thiobacillus, Escherichia, and Kushneria (Ouahmane et al., 2007; Jha et al., 2009; Selvakumar et al., 2011; Zhu et al., 2011). Here we demonstrate that the bacterial strains isolated from alkaline, dark, and oligotrophic karst caves can also dissolve zinc phosphate. This expands our knowledge about the habitats of PSB in natural environments. It is not surprising that P. fluorescens P35 and P. poae P41 can solubilize zinc phosphate with an efficiency of 16.7% and 17.6%, respectively, based on the calculation of soluble Zn concentration. Furthermore, we also isolated a strain identified as E. aurantiacum E11 which is capable of zinc phosphate dissolution with a higher efficiency of 39.7%. To our knowledge, it is reported nowhere else about the capability of E. aurantiacum E11 to solubilize phosphorus minerals. This indicates that solubilizing P-minerals might be a universal activity of heterotrophs.

Among the reported PSB genera, Pseudomonas is the most studied one. The reported dissolution efficiency varied from 0.95%-26.2% for Ca₃(PO₄)₂ (Illmer and Schinner.1992; Hu et al., 2010; Li et al.,2010; Govindan et al., 2011). The dissolution efficiency of Zn₃(PO₄)₂ was reported to be 47% by P. fluorescens 3a (Di Simine et al.,1998), which is higher than that by E. aurantiacum E11 isolated in our work. However, the dissolution efficiencies of Zn₃(PO₄)₂ by P. fluorescens P35 and P. poae P41 are lower than that by E. aurantiacum E11(39.7%) in our own experiments. The efficiency might be affected by carbon source, pH, temperature, the concentrations of sodium chloride and glucose. It was suggested that optimal dissolution condition for Zn₃(PO₄)₂ by P. fluorescens 3a is at 20°C, and pH 7, with glucose as the carbon source (Di Simine et al.,1998). Our experiments were conducted at 30°C and at an initial pH of 6.2 with glucose as the sole carbon source. It is likely that E. aurantiacum E11 isolated from the cave might dissolve Zn₃(PO₄)₂ with higher efficiency under the optimal conditions. Consequently, E. aurantiacum E11 may play a significant role on the phosphorus release in some extreme environments as observed in our work. Given that the released Zn(II) might occur as secondary precipitate or adsorb on the cell surface, the actual dissolution efficiency of zinc phosphate should be higher than the value we reported.

Microbial dissolution mechanism

Different mechanisms have been proposed to explain the PSB dissolution of P-minerals; they include the releasing of organic acids, phosphatases, chelating compounds, mineral acids and siderophores (Gaskins et al., 1985; Schippers and Chanway, 1987; Podile and Dube, 1988; Singh et al., 1989; Kim et al., 1998; Landeweert et al., 2001; Vassilev et al., 2001; Pradhan and Sukla, 2005). All of these mechanisms proposed are related to the production of PSB metabolite which can result in the decrease of environmental pH. For example, bacteria can produce various organic acids via different metabolic pathways (Lin et al., 2006; Mardad et al., 2013) to decrease the environmental pH. As a result, the level of soluble phosphorus released was positively correlated with the concentration of viable bacteria (Chen et al., 2008).

The pH values measured in our experiments decreased from 6.2 to 4.51 or even lower in the inoculated systems. The aqueous Zn(II) concentration was positively correlated with H⁺ activity, suggesting that pH played a key role in solubilization of Zn₃(PO₄)₂. Even though we did not determine the specific organic acids produced throughout the whole experimental course, the chemical control experiments with the addition of inorganic acid (HCl) further confirmed the importance of acidification in Zn₃(PO₄)₂ dissolution.

Bacterial dissolution can occur via indirect and direct contact with minerals. By direct contact, bacteria can attach to the surface of minerals, leaving abundant itching pitches when mineral dissolution occurs (Lu and Wang, 2012; Liu et al., 2011). Few itching pitches were observed by SEM in the residues collected in our experiments, inferring that microbes are not necessary to directly attach to the surface of Zn₃(PO₄)₂ during the dissolution.

Biogeochemical and environmental implication

Due to the thick overlying rocks (500-600 m) and the potential microbial activities in the long transportation of groundwater, most photosynthetic organic matter derived from the overlaid soil might be consumed before reaching the Heshang cave. The organic matter transported into the cave is predominantly refractory, which constitutes the dominant organic matter to sustain the subsurface heterotrophic biota. In addition to the organic matter, phosphorus might be one of the key limiting factors for both heterotrophic and autotrophic microbial growth under the alkaline condition due to the low solubility of P-containing minerals. The isolation of the three PSB strains from the alkaline, dark, and oligotrophic Heshang cave strongly suggests that some bacteria in dripping water may play an important role in sustaining the dark ecosystem by offering soluble phosphorus. Our results greatly enhance our understanding of the ecosystem function in dark caves.

The increase of Zn concentration in the solution appears to cause no inhibition to microbial growth as indicated by the spectrophotometer measurements in our work. Previously, it was demonstrated that Zn(II) was toxic to various microbes in overdoses (McGrath et al. 1995; Di Simine et al., 1998; Nweke et al., 2007). For Bacillus sp. SED1 and Arthrobacter sp. SED4 isolated from the fluvial sediment at Choba, Zn(II) would cause an inhibition of 50% activity of dehydrogenase with a concentration ranging from 13.47±1.96 to 52.79±4.32 mg/L, and a complete inhibition of dehydrogenase activity with the concentration ranging from 78.43±2.73 to 94.32±4.06 mg/L (Nweke et al., 2007). Strains isolated from a long-term Zn contaminated area in a Hungarian (Nagyhörcsök) experimental field can tolerant 75 and 100 mg/L Zn(II) (Vivas et al., 2006). The viable counts of P. fluorescens 3a started to decrease at the Zn concentration around 98.12 mg/L, followed by a sharp decrease at 228.94 mg/L indicating the Zn toxicity (Di Simine et al., 1998). However, the protein content kept increasing throughout the experimental course, contradictory to the viable counts (Di Simine et al., 1998). It is difficult to make an assessment about the Zn toxicity to our isolates due to unavailable viable counts in our experiment, which awaits further investigation. Nevertheless, it is well accepted that an over exposure to Zn(II) will have negative effects on microbes as well as on humans.

Conclusions

As an important nutrient carrier in oligotrophic karst caves, dripping water is found to harbor some phosphorus solubilizing bacteria identified to be E. aurantiacum E11, P. fluorescens P35 and P. poae P41 in Heshang cave in central China. Here we reported E. aurantiacum E11 is able to solubilize P-minerals with much higher dissolution efficiency than P. fluorescens P35 and P. poae P41, two species of the most studied genus, Pseudomonas. The correlation between H⁺ activity and released aqueous Zn(II) concentration demonstrated the presence of PSB acidification during the zinc phosphate dissolution. This is further supported by the few itching pitches on zinc phosphate particles shown by SEM images. No inhibition was observed on the microbial growth even when the soluble Zn(II) reached to 370 mg/L in E. aurantiacum E11 inoculated system. Our work on the bacterial isolates from extreme environments expands our knowledge about the natural habitats of PSB in addition to soils and sediments reported before, and sheds light on the understanding of the microbially-mediated elemental geochemical cycles in karst caves.

References

[1] Atmaca S, Gül K, Cicek R (1998). The effect of zinc on microbial growth. Tr J of Medical Sciences, 28: 595–597

[2] Bidle K D, Lee S, Marchant D R, Falkowski P G (2007). Fossil genes and microbes in the oldest ice on Earth. PNAS, 104(33):13455–13460

[3] Bong C W, Malfatti F, Azam F, Obayashi Y, Suzuk S (2010). The effect of zinc exposure on the bacteria abundance and proteolytic activity in seawater. In: Hamamura N, Suzuki S, Mendo S, Barroso C M, Iwata H, Tanabe S, eds. Interdisciplinary Studies on Environmental Chemistry — Biological Responses to Contaminants. Tokyo: Terrapub, 57–63

[4] Chelius M K, Beresford G, Horton H, Quirk M, Selby G, Simpson R T, Horrocks R, Moore J C (2009). Impacts of alterations of organic inputs on the bacterial community within the sediments of Wind Cave, South Dakota, USA. Int J Speleol, 38(1): 1–10

[5] Chen Z X, Ma S W, Liu L L (2008). Studies on phosphorus solubilizing activity of a strain of phosphobacteria isolated from chestnut type soil in China. Bioresour Technol, 99(14): 6702–6707

[6] Cunninghum J E, Kuiack C (1992). Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii. Appl Environ Microbiol, 58(5): 1451–1458Please See page 2 line 8).

[7] Di Simine C D, Sayer J A, Gadd G M (1998). Solubilization of zinc phosphate by a strain of Pseudomonas fluorescens isolated from a forest soil. Biol Fertil Soils, 28(1): 87–94

[8] Engel A S (2010). Microbial diversity of cave ecosystems. In: Alexander L, Martin M, Barton L L, eds. Geomicrobiology: Molecular and Environmental Perspective. Dordrecht Heidelberg London New York: Springer, 219–238

[9] Fasim F, Ahmed N, Parsons R, Gadd G M (2002). Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery. FEMS Microbiol Lett, 213(1): 1–6

[10] Feng K, Lu H M, Sheng H J, Wang X L, Mao J (2004). Effect of organic ligands on biological availability of inorganic phosphorus in Soils. Pedosphere, 14(1): 85–92

[11] Gaskins M H, Albrecht S L, Hubbell D H (1985). Rhizosphere bacteria and their use to increase plant productivity. Agr Ecosyst Environ , 12(2): 99–116

[12] Govindan S, Piyush J, Preeti S (2011). Pseudomonas lurida M2RH3 (MTCC 9245), a psychrotolerant bacterium from the Uttarakhand Himalayas, solubilizes phosphate and promotes wheat seedling growth. World J Microbiol Biotechnol, 27(5): 1129–1135

[13] Hu C, Henderson G M, Huang J, Chen Z H, Johnson K R (2008). Report of a three-year monitoring programme at Heshang cave, central China. Int J Speleol, 37: 143–151

[14] Hu X J, Li Z J, Cao Y C, Zhang J, Gong Y X, Yang Y F (2010). Isolation and identification of a phosphate-solubilizing bacterium Pantoea stewartii subsp.stewartii g6, and effects of temperature, salinity, and pH on its growth under indoor culture conditions. Aquacult Int, 18(6): 1079–1091

[15] Hughes M N, Poole R K (1989). Metals and microorganisms. London: Chapman and Hall

[16] Illmer P, Schinner F (1992) Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biolol Biochem, 24: 389–395

[17] Jha B K, Gandhi Pragash M, Cletus J, Raman G, Sakthivel N(2009). Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonas strains, Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii. World J Microbiol Biotechnol, 25(4): 573–581

[18] Kang S C, Ha C G, Lee T G, Maheshwari D K (2002). Solubilisation of insoluble inorganic phosphates by soil-inhabiting fungus Fomitopsis sp. PS 102. Curr Sci, 82(4): 439–442

[19] Khan M S, Zaidi A, Wani P A (2007). Role of phosphate-solubilizing microorganisms in sustainable agriculture-a review. Agron sustaine Dev, 27(1): 29–43

[20] Kim K Y, Jordan D, McDonald G A (1998). Enterobacter agglomerans, phosphate solubilizing bacteria and microbial activity in soil: effect of carbon sources. Soil Biol Biochem, 30(8-9): 995–1003

[21] Landeweert R, Hoffland E, Finlay R D, Kuyper T W, van Breemen N (2001). Linking plants to rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol, 16(5): 248–254

[22] Lee N M, Meisinger D B, Aubrecht R, Kovacikfi L, Porter M L, Engel A S (2012). Caves and Karst Environments. In: Bell E ed. Life at Extremes: in Environments, Organisms and Strategies for Survival, CABI, 320–344

[23] Li X T, Dong C X, Yang X M, Zhong Z T, Shen Q R, Xu Y C (2010). Labeling of phosphate-solubilizing bacteria K3 with GFP and its phosphate solulilization ability. Soils, 42(4): 548–553 (in Chinese)

[24] Lin T F, Huang H I, Shen F T, Young C C (2006). The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-Al74. Bioresour Technol, 97: 957–960

[25] Liu D, Wang H M, Dong H L, Qiu X, Dong X Z, Cravotta C A III (2011). Mineral transformations associated with goethite reduction by Methanosarcina barkeri. Chem Geol, 288(1-2): 53–60

[26] Liu Q Y, Wang H M, Zhao R, Gong L F (2010). Bacteria isolated from dripping water in the oligotrophic Heshang cave in central China. J Earth Sci, 21(S1): 325–328

[27] López-Bucio J, de La Vega O M, Guevara-García A, Herrera-Estrella L (2000). Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nat Biotechnol, 18(4): 450–453

[28] Lu X C, Wang H M (2012). Microbial oxidation of sulfide tailings and the environmental consequences. Elements, 8(2): 119–124

[29] Mardad I, Serrano A, Soukri A (2013). Solubilization of inorganic phosphate and production of organic acids by bacteria isolated from a Moroccan mineral phosphate deposit. Afri J Microbiol Res, 7(8): 626-635

[30] McGrath S P, Chaudri A M, Giller K E (1995). Long-term effects of metals in sewage sludge on soils, microorganisms and plants. J Ind Microbiol, 14(2): 94–104

[31] Mikonova O, Novakova J (2002). Evaluation of P-solubilising activity of soil microorganisms and its sensitivity to soluble phosphate. Rostlinna Vyroba, 48(9): 397–400

[32] Nweke C O, Alisi C S, Okolo J C, Nwanyanwu C E (2007). Toxicity of zinc to heterotrophic bacteria from a tropical river sediment. Appl Eco Environ Res, 5(1): 123–132

[33] Ouahmane L, Thioulouse J, Hafidi M, Prin Y, Ducousso M, Galiana A, Plenchette C, Kisa M, Duponnois R (2007). Soil functional diversity and P solubilization from rock phosphate after inoculation with native or allochtonous arbuscular mycorrhizal fungi. For Ecol Manage, 241(1-3): 200–208

[34] Peix A, Rivas-Boyero A A, Mateos P F, Rodirguez-Barrueco C, Martinez-Molina E, Velazquez E (2001). Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biol Biochem, 33(1): 103–110

[35] Pérez E, Sulbarán M, Ball M M, Yarzábal L A (2007). Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region. Soil Biol Biochem, 39(11): 2905–2914

[36] Podile A R, Dube H L (1988). Plant growth-promoting activity of Bacillus subtilis AF1. Curr Sci, 57(4): 183–186

[37] Pradhan N, Sukla L B (2005). Solubilisation of inorganic phosphate by fungi isolated from agriculture soil. Afr J Biotechnol, 5(10): 850–854

[38] Rosling A, Suttle K B, Johansson E, van Hees P A W, Banfield J F (2007). Phosphorous availability influences the dissolution of apatite by soil fungi. Geobiology, 5(3): 265–280

[39] Saravanan V S, Madhaiyan M, Thangaraju M (2007). Solubilization of zinc compounds by the diazotrophic, plant growth promoting bacterium Gluconacetobacter diazotrophicus. Chemosphere, 66(9): 1794–1798

[40] Schippers B, Bakker A W, Bakker P A H M (1987). Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Ann Rev Phytopathol25: 339–358

[41] Selvakumar G, Joshi P, Suyal P, Mishra P K, Joshi G K, Bisht J K, Bhatt J C, Gupta H S(2011). Pseudomonas lurida M2RH3 (MTCC 9245), a psychrotolerant bacterium from the Uttarakhand Himalayas, solubilizes phosphate and promotes wheat seedling growth. World J Microbiol Biotechnol, 27(5): 1129–1135

[42] Singh J S, Raghabanshi A S, Singh R S, Srivastava S C (1989). Microbial biomass acts as a source of plant nutrients in dry tropical forest and savanna. Nature, 338(6215): 499–500

[43] Singh M S, Yadav L S, Singh S K, Singh P (2011). Phosphate solubilizing ability of two Arctic Aspergillus niger strains. Polar Res, 30: 7283

[44] Tao G C, Tian S J, Cai M Y, Xie G H (2008). Phosphate-solubilizing and mineralizing abilities of bacteria isolated from soils. Pedosphere, 18(4): 515–523

[45] Vassilev N, Vassileva M, Fenice M, Federici F (2001). Immobilized cell technology applied in solubilization of insoluble inorganic (rock) phosphates and P plant acquisition. Bioresour Technol, 79(3): 263–271

[46] Vivas A, Biró B, Ruíz-Lozano J M, Barea J M, Azcón R (2006). Two bacterial strains isolated from a Zn-polluted soil enhance plant growth and mycorrhizal efficiency under Zn-toxicity. Chemosphere, 62(9): 1523–1533

[47] Welch S A, Taunton A E, Banfield J F (2002). Effect of microorganisms and microbial metabolites on apatite dissolution. Geomicrobiol J, 19(3): 343–367

[48] Xiang W L, Liang H Z, Liu S, Luo F, Tang J, Li M, Che Z (2011). Isolation and performance evaluation of halotolerant phosphate solubilizing bacteria from the rhizospheric soils of historic Dagong Brine Well in China. World J Microbiol Biotechnol, 27(11): 2629–2637

[49] Zhu F L, Qu L Y, Hong X G, Sun X Q (2011). Isolation and characterization of a phosphate-solubilizing halophilic bacterium Kushneria sp. YCWA18 from Daqiao Saltern on the Coast of Yellow Sea of China. Evidence-Based Complementary and Alternative Medicine, 2011,

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