ClockworkOrange
+SATOR AREPO TENET OPERA ROTAS+
well this one is a pretty good read. I too couldnt D/L the PDF. i did some time back.ill poke around and see if I can find it. not sure though,LOL! but this one is large but tons of info in the first half of the paper.
Phosphate Solubilization: Their Mechanism Genetics And Application
N Ahmed, S Shahab
Citation
N Ahmed, S Shahab. Phosphate Solubilization: Their Mechanism Genetics And Application. The Internet Journal of Microbiology. 2009 Volume 9 Number 1.
Abstract
The global necessity to increase agricultural production from a steadily decreasing and degrading land resource base has placed considerable strain on agro ecosystems (Tilak, 2005). Current strategy is to maintain and improve agricultural productivity exclusively via the use of chemical fertilizers. Although the use of chemical fertilizers is credited with nearly fifty percent increase in agricultural production but they are closely associated with environmental pollution and health hazards (Gaur and Gaind, 1999). Many synthetic fertilizers contain acids, such as sulfuric acid and hydrochloric acid, which tend to increase the acidity of the soil, reduce the soil's beneficial organism population and interfere with plant growth. Generally, healthy soil contains enough nitrogen-fixing bacteria to fix sufficient atmospheric nitrogen to supply the needs of growing plants. However, continued use of chemical fertilizers may destroy these nitrogen-fixing bacteria. Furthermore, chemical fertilizers may affect plant health. For example, citrus trees tend to yield fruits that are lower in vitamin C when treated with synthetic fertilizer. Lack of trace elements in soil regularly dosed with chemical fertilizers is not uncommon. This lack of vital micronutrients can generally be attributed to the use of chemical fertilizers. On the other hand Biofertilizer adds nutrients to soil.Environmentally friendly biotechnological approaches offer alternatives to chemical fertilizers (Dobbelaere et al., 2003). Given the negative environmental impacts of chemical fertilizers and their increasing costs, the use of PGPB is thus being considered as an alternative or a supplemental way of reducing the use of chemicals in agriculture (De Weger et al., 1995; Gerhardson, 2002, Postma, et al., 2003; Welbaum, 2004)
Introduction
The global necessity to increase agricultural production from a steadily decreasing and degrading land resource base has placed considerable strain on agro ecosystems (Tilak, 2005). Current strategy is to maintain and improve agricultural productivity exclusively via the use of chemical fertilizers. Although the use of chemical fertilizers is credited with nearly fifty percent increase in agricultural production but they are closely associated with environmental pollution and health hazards (Gaur and Gaind, 1999). Many synthetic fertilizers contain acids, such as sulfuric acid and hydrochloric acid, which tend to increase the acidity of the soil, reduce the soil's beneficial organism population and interfere with plant growth. Generally, healthy soil contains enough nitrogen-fixing bacteria to fix sufficient atmospheric nitrogen to supply the needs of growing plants. However, continued use of chemical fertilizers may destroy these nitrogen-fixing bacteria. Furthermore, chemical fertilizers may affect plant health. For example, citrus trees tend to yield fruits that are lower in vitamin C when treated with synthetic fertilizer. Lack of trace elements in soil regularly dosed with chemical fertilizers is not uncommon. This lack of vital micronutrients can generally be attributed to the use of chemical fertilizers. On the other hand Biofertilizer adds nutrients to soil.
Environmentally friendly biotechnological approaches offer alternatives to chemical fertilizers (Dobbelaere et al., 2003). Given the negative environmental impacts of chemical fertilizers and their increasing costs, the use of PGPB is thus being considered as an alternative or a supplemental way of reducing the use of chemicals in agriculture (De Weger et al., 1995; Gerhardson, 2002, Postma, et al., 2003; Welbaum, 2004)
It has been estimated that in some soil up to 75% of applied phosphate fertilizer may become unavailable to the plant because of mineral phase reprecipitation (Goldstein, 1986; Sundara et al., 2002). Phosphate-solubilizing bacteria (PSB) are able to convert insoluble phosphates into soluble forms (Illmer and Schinner, 1995; Hilda et al., 2000ab; Peix et al., 2001 ab; Viverk and Singh, 2001; Sudhakara et al., 2002) and have therefore been used to enhance the solubilization of reprecipitated soil P for crop improvement (Shekhar et al., 2000; Young et al., 1986; Young, 1990).
Phoshate Availability in Soil
Phosphorus (P) is one of the major essential macronutrients for biological growth and development (Ehrlich, 1990). It is present at levels of 400–1200 mg/kg of soil (Fernandez, 1988). The concentration of soluble P in soil is usually very low, normally at levels of 1 ppm or less then 1ppm (Goldstein, 1994). The cell might take up several P forms but the greatest part is absorbed in the forms of Phosphate (Beever and Burns, 1980).
Figure 1
Figure 1: Phosphate cycle (
Mineral forms of phosphorus are represented in soil by primary minerals, such as apatite, hydroxyapatite, and oxyapatite. They are found as part of the stratum rock and their principal characteristic is their insolubility. In spite of that, they constitute the biggest reservoirs of this element in soil because, under appropriate conditions, they can be solubilized and become available for plants and microorganisms. Mineral phosphate can be also found associated with the surface of hydrated oxides of Fe, Al, and Mn, which are poorly soluble and assimilable. This is characteristic of ferralitic soils, in which hydration and accumulation of hydrated oxides and hydroxides of Fe takes place, producing an increase of phosphorus fixation capacity (Fernandez, 1988).
There are two components of P in soil, organic and inorganic phosphates. A large proportion is present in insoluble forms, and therefore, not available for plant nutrition. Inorganic P occurs in soil, mostly in insoluble mineral complexes, some of these appearing after the application of chemical fertilizers. These precipitated forms cannot be absorbed by plants. Organic matter, on the other hand, is an important reservoir of immobilized P that accounts for 20–80% of soil P (Richardson, 1994).
Organic Phosphate
A second major component of soil P is organic matter. Organic forms of P may constitute 30–50% of the total phosphorus in most soils, although it may range from as low as 5% to as high as 95% (Paul and Clark, 1988). Organic P in soil is largely in the form of inositol phosphate (soil phytate). It is synthesized by microorganisms and plants and is the most stable of the organic forms of phosphorus in soil, accounting for up to 50% of the total organic P (Dalal, 1977; Anderson 1980; Harley and Smith, 1983). Other organic P compounds in soil are in the form of phosphomonoesters, phosphodiesters including phospholipids and nucleic acids, and phosphotriesters.Of the total organic phosphorus in soil, only approximately 1% can be identified as nucleic acids or their derivatives (Paul and Clark, 1988). Various studies have shown that only approximately 1–5 ppm of phospholipids phosphorus occurs in soil, although values as high as 34 ppm have been detected (Paul and Clark, 1988). Large quantities of xenobiotic phosphonates, which are used as pesticides, detergent additives, antibiotics, and flame retardants, are released into the environment. These C-P compounds are generally resistant to chemical hydrolysis and biodegradation, but several reports have documented microbial P release from these sources (Ohtake, 1996; McGrath, 1998).
Figure 2
Figure 2: Figure showing complexity of average soil
(http:/ www.physicalgeography.net/fundamentals/10t.html)
Organic Phosphate Solubilization
Organic phosphate solubilization is also called mineralization of organic phosphorus, and it occurs in soil at the expense of plant and animal remains, which contain a large amount of organic phosphorus compounds. The decomposition of organic matter in soil is carried out by the action of numerous saprophytes, which produce the release of radical orthophosphate from the carbon structure of the molecule. The organophosphonates can equally suffer a process of mineralization when they are victims of biodegradation (McGrath, 1995). The microbial mineralization of organic phosphorus is strongly influenced by environmental parameters; in fact, moderate alkalinity favors the mineralization of organic phosphorus (Paul and Clark, 1988) The degradability of organic phosphorous compounds depend mainly on the physicochemical and biochemical properties of their molecules, e.g. nucleic acids, phospholipids, and sugar phosphates are easily broken down, but phytic acid, polyphosphates, and phosphonates are decomposed more slowly (Ohtake, 1996; McGrath, 1995; McGrath 1998).
Phosphorus can be released from organic compounds in soil by three groups of enzymes:
Nonspecific phosphatases, which perform dephosphorylation of phospho-ester or phosphoanhydride bonds in organic matter
Phytases, which specifically cause P release from phytic acid
Phosphonatases and C–P Lyases, enzymes that perform C–P cleavage in organophosphonates
The main activity apparently corresponds to the work of acid phosphatases and phytases because of the predominant presence of their substrates in soil.
Figure 3
Figure 3: Mineralization of organic compounds within soil
(Source:http://grunwald.ifas.ufl.edu/Nat_resources/organic_matter/som.gif)
Inorganic Phosphate Mineralization
Several reports have suggested the ability of different bacterial species to solubilize insoluble inorganic phosphate compounds, such as tricalcium phosphate, dicalcium phosphate, hydroxyapatite, and rock phosphate (Goldstein, 1986). In two thirds of all arable soils, the pH is above 7.0, so that most mineral P is in the form of poorly soluble calcium phosphates (CaPs). Microorganisms must assimilate P via membrane transport, so dissolution of CaPs to Pi (H2PO4) is considered essential to the global P cycle. Evaluation of samples from soils throughout the world has shown that, in general, the direct oxidation pathway provides the biochemical basis for highly efficacious phosphate solubilization in Gram-negative bacteria via diffusion of the strong organic acids produced in the periplasm into the adjacent environment. Therefore, the quinoprotein glucose dehydrogenase (PQQGDH) may play a key role in the nutritional ecophysiology of soil bacteria. MPS bacteria may be used for industrial bioprocessing of rock phosphate ore (a substituted fluroapatite) or even for direct inoculation of soils as a ‘biofertilizer’ analogous to nitrogen-fixing bacteria. Both the agronomic and ecological aspects of the direct oxidation mediated MPS trait. (Gold stein et al., 2003)
Among the bacterial genera with this capacity are Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Microccocus, Aereobacter, Flavobacterium and Erwinia (Babu-khan et al 1995; Goldstein, 1987; Sperber 1958; Rodríguez and Fraga, 1999).
Mechanism of Phosphate Solubilization
A number of theories have been proposed to explain the mechanism of phosphate solubilization. Important among them are:
Acid production theory
Proton and enzyme theory
Acid Production Theory
According to this theory, the process of phosphate solubilization by PSM is due to the production of organic acids which is accompanied by the acidification of the medium (Puente et al., 2004). A decrease in the pH of the filtrate from the initial value of 7.0 to a final value of 2.0 was recorded by many workers (Gaur and Sachar 1980; Gaind and Gaur 1990, 1991; Illmer and Schinner, 1992). The analysis of culture filtrates of PSMs has shown the presence of number of organic acids such as malic, glyoxalic, succinic, fumaric, tartaric, alpha keto butyric, oxalic, citric, 2-ketogluconic and gluconic acid (Lapeyrie et al., 1991; Cuningham and kuaick, 1992; Ilmer and schiner, 1995; Fasim et al., 2002, Gadd 1997; Kim et al., 1997)
The amount and type of the organic acid produced varied with the microorganism. The organic acids released in the culture filtrates react with the insoluble phosphate. The amount of soluble phosphate released depends on the strength and type of acid. Aliphatic acids are found to be more effective in P solubilization then phenolic acids and citric acids. Fumaric acid has highest P solubilizing ability. Tribasic and dibasic acids are also more effective than monobasic acids. In the presence of tribasic acids and dibasic acids, a secondary effect appears due to ability of these acids to form unionized association compounds with calcium thereby removing calcium from the solution and increasing soluble phosphate concentration (Gaur and Gaind,1999).
Organic acids contribute to the lowering of solution pH as they dissociate in a pH dependent equilibrium, into their respective anion(s) and proton(s). Organic acids buffer solution pH and will continue to dissociate as protons are consumed by the dissolution reaction (Welch et al., 2002). Similarly, microorganisms often export organic acids as anions (Duro and Serrano 1981, Konings 1985, Netik et al., 1997).
Besides organic acids, inorganic acids such as nitric and sulphuric acids are also produced by the nitrifying bacteria and thiobacillus during the oxidation of nitrogenous or inorganic compounds of sulphur which react with calcium phosphate and convert them into soluble forms (Gaur and Gaind, 1999).
The most efficient mineral phosphate solubilization (MPS) phenotype in Gram negative bacteria results from extracellular oxidation of glucose via the quinoprotein glucose dehydrogenase to gluconic acid (Kpomblekou and Tabatabai, 1994; Hilda and Fraga, 1999; Hilda et al., 2000). The resulting pH change and reduction potential are thought to be responsible for the dissolution of phosphate in the culture medium.
Figure 4
Figure 4: Production of gluconic acid via the alternative extracellular oxidation pathway of glucose metabolism.
(Source: http://www.ucc.ie/biomerit/simon image.gif)
Gluconic acid biosynthesis is carried out by the glucose dehydrogenase (GDH) enzyme and the co-factor, pyrroloquinoline quinone (PQQ). Goldstein and Liu (1987) cloned a gene from Erwinia herbicola that is involved in mineral phosphate solubilization. The expression of this gene allowed production of gluconic acid and mineral phosphate solubilization activity in E.coli HB101.
Gluconic acid is the principal organic acid produced by Pseudomonas sp. (Illmer and Schinner, 1992), Erwinia herbicola (Liu et al., 1992) Pseudomonas cepacia (Goldstein et al., 1994) and Burkholderia cepacia (Rodríguez and Fraga 1999) Rhizobium leguminosarum (Halder et al., 1990) Rhizobium meliloti (Halder and Chakrabartty, 1993) and Bacillus firmus (Banik and Dey, 1982) produce noticeable amounts of 2-ketogluconic acid. Fasim et al., (2002) have reported bacterial solubilization of insoluble zinc oxide and zinc phosphate, mediated by the production of gluconic and 2-ketogluconic acid. Other organic acids, such as lactic, isovaleric, isobutyric, acetic, glycolic, oxalic, malonic and succinic acids are also generated by different phosphate solubilizing bacteria (Rodríguez and Fraga 1999).
Goebel and Krieg (1984) showed that gluconic acid was not formed during growth of either A. brasilense or A. lipoferum on fructose (a common carbon source for both), and was detected only during growth of glucose. Rodríguez (2004) reported that A. brasilense can produce gluconic acid in vitro when grown on fructose and amended with glucose as an inducer for gluconic acid production and have in vitro phosphate solubilizing capability.
Glucose is the precursor for synthesis of gluconic acid (Rodrigues etal 2004). This has suggested that Phosphate solubilization in these strains is mediated by glucose or gluconic acid metabolism. As solubilization of phosphate preceded detection of gluconic acid in the medium, perhaps even low levels of the acid (below the detection level of HPLC) started to dissolve the sparingly soluble phosphate. Alternatively, consumption of gluconic acid by growing cells could also take place. In A. brasilense, reduction in the quantity of soluble phosphate after incubation for 48 h can be explained as auto consumption of soluble phosphate by the growing bacterial population (Rodriguez et al., 2000).
The latter may result from production of gluconic acid and NH4 + uptake, which may release protons to the medium. In the faster growing A. brasilense strains, perhaps the cells used more NO-3 at the end of the incubation time, thereby releasing OH-, which may account for the higher pH after 48 h. The metabolic mechanism by which gluconic acid was produced was not explored (Rodríguez et al., 2004)
The P-solubilizing capability of gluconic acid was much higher as compared to 2-keto-gluconic acid in the filtrate from strain CC-Al74 culture. The process of acidification and chelation by gluconic acid and 2-keto gluconic acid dissolved tri calcium phosphate (TCP) in cultural medium. The chelation property of gluconic acid enables it to form insoluble complex. Insoluble metal forms may be solubilized by protons, with Ca++ liberating phosphates (Kpomblekou and Tabatabai, 1994; Reyes et al., 1999; Shekhar et al., 2000).
Protons can be pumped into the external medium by various membrane associated pumps which set up ionic gradients for the acquisition of nutrients (Jones and Gadd, 1990; Sigler, and Hofer, 1991; Gadd, 1993). In addition, protons arise from produced organic acids which also possess an organic acid anion which is usually capable of forming a complex with metal cation (Burgstaller,. and Schinner, 1993; Hughes and Poole, 1991).
The production of citric or gluconic acid and the extrusion of H+ result from membrane transport mechanisms was described as possible mechanism for dissolving rock-phosphate from hydroxy apatite, iron phosphate, and aluminum phosphate by Penicillium rugulosum (Reyes et al., 1999). These processes are influenced by the sources of the nitrogen, phosphate, and carbon. Citric acid production and the resulting amount of phosphate dissolution are increased if nitrate is the only nitrogen source. Because citric acid is involved not only in the dissolution of phosphate but also in dissolution of iron and other metals from minerals, the process of nitrate accumulation in soils might play an important role for the weathering of rock in general.
The nature and type of acid production is mainly dependent on the carbon source (Reyes et al., 1999). In general, oxalic, citric, and gluconic acid, are strong solubilizing agents of feldspar, biotite, and phyllosilicates, (Torre et al., 1993)
Figure 5
Table 1: Gluconic acid production by various bacterial strains
Proton and Enzyme Theory
Esterase type enzymes are known to be involved in liberating phosphorus from organic phosphatic compounds. PSMs (phosphate solubilizing microorganisms) are also known to produce phosphatase enzyme along with acids which cause the solubilization of P in aquatic environment (Alghazali et al., 1986). Illmer and Schinner (1995) reported that out of the four efficient phosphates solubilizing microbes, Penicillium aurathiogriseum, Penicilllium simplicissimum, Aspergillus niger and Pseudomonas sp only A .niger could produce orgainic acid. Two most probable explanations for this are:
Solubilization without acid production is due to the release of protons accompanying respiration or ammonium assimilation (Taha et al., 1969; Kucey 1983; Dighton and Boddy 1989; Parks et al., 1990)
More solubilization occurs with ammonium salts than with nitrate salts as the nitrogen source in the media (Gaur and Gaind, 1999).
Besides these two mechanisms the production of chelating substances (Luo et al., 1993) H2S, CO2 (Kapoor et al., 1989) mineral acids, siderophores (Bossier et al., 1988) biologically active substances like indole acetic acids, gibberllines and cytokinins (Kucey et al., 1988) are also correlated with Phosphate solubilization. Chelation involves the formation of two or more coordinate bonds between an anionic or polar molecule and a cation, resulting in a ring structure complex (Whitelaw, 2000). Organic acid anions, with oxygen containing hydroxyl and carboxyl groups, have the ability to form stable complexes with cations such as Ca2+, Fe2+, Fe3+, and Al3+, that are often bound with phosphate in poorly forms (Jones ,1998 ; Kucey 1988)
Dissolution of phosphate in soil is a very important process for plant growth. Several studies have shown that the phosphate uptake by plants can be markedly increased by either mycorrhizal fungi (Azcon-Aguilar et al., 1986) or inoculation of soil with species capable of solubilizing free phosphate, such as P. Bilaii (Cunningham and Kuiack 1992; Uzair et al., 2006).
Phosphate –Plant Interaction
Phosphorus is one of one of the major plant nutrient limiting plant growth. It plays a key role in nutrition of plants as it promotes development of deeper roots. The average soil is rich in phosphorus as it contains about 0.05% (w/w) phosphorus (Barber, 1984) but only one tenth of this is available to plants approximately 95–99% is present in the form of insoluble phosphates and hence cannot be utilized by the plants and due to its poor solubility and chemical fixation in the soil (Gaurand Gaind, 1999) causing a low efficiency of soluble P fertilizers.
To increase the availability of phosphorus for plants, large amounts of fertilizer is used on a regular basis. But after application, a large proportion of fertilizer phosphorus is quickly transferred to the insoluble forms. Therefore, very little percentage of the applied phosphorus is used, making continuous application necessary. (Abd Alla, 1994).
Figure 6
Figure 5: Soil and root interactions
(http://www.sare.org/publications/bsbc/fig3_3.jpg)
Soils microorganisms are involved in a range of processes that affect Phosphate transformation and thus influence the subsequent availability of phosphate to plant roots (Richardson, 2001). Free living phosphate solubilizing microorganisms (PSM) are always present in soils. The populations of inorganic Phosphate solubilizing microorganisms are sometimes very low, less than 102 CFU g-1 of soil as observed in a soil in Northern Spain (Peix et al., 2001). In four Quebec soils the number of root free PSM ranged from 2.5-to 3 x 106 CFU g-1 of soil and they represented from 26- 46% of the total soil microflora (Chabot et al., 1993). As observed with other soil microbes the number of PSM is more important in the rhizosphere than in non rhizosphere soil (Kucey et al., 1989), and the number of phosphate solubilizing bacteria is more important than that of fungi (Kucey, 1983). However, inoculation studies aimed to improving P nutrition in plants involved bacteria and fungi, and is commercially available in Western Canada as the phosphate inoculant Jumpstart (Philom Bios, Saskatoon, Sask.). They are sold for wheat, canola, mustard and other legumes and contain an bacterial strain of Penicillium Bilaii. (http://www.philombios.ca/).
Plant Growth Promoting Bacteria
Although plant growth promoting bacteria occur in soil, usually their numbers are not high enough to compete with other bacteria commonly established in the rhizosphere. Therefore, for agronomic utility, inoculation of plants by target microorganisms at a much higher concentration than those normally found in soil is necessary to take advantage of their beneficial properties for plant yield enhancement. (Igual, 2001)
Figure 7
Figure 6: Microbial activities in the soil for plant growth promotion.
(Source: http://www.treepower.org/soils/soil-benefits.jpg)
In recent years, interest in soil microorganisms that can promote plant growth has been increased considerably. The use of PGPRs to control soil borne pathogens is a practice with a promising future, because the Montreal Protocol (an international treaty to protect the earth from the detrimental effects) proposes the elimination of toxic chemicals. This has forced the plant scientists to look for new alternatives to replace fertilizers. A number of different bacteria have been reported to promote plant growth, including Azotobacter sp., Azospirillum sp., Pseudomonas sp., Acetobacter sp., Burkholderia sp. and Bacillus sp. (Rodrigues and Fraga 1999)
This one i downloaded. Only the asian stuff wasnt working, but i'll have to work me through this one first, anyway.
