Future Trends in Research on Microbial Phosphate Solubilization: Moving Beyond One Hundred Years of Insolubility

Alan H. Goldstein

Fierer Chair and Director, Center for Biomaterials Research, Alfred University, 2 Pine Street , Alfred, New York, 14802 USA

Phone (607) 871-2645, FAX (607) 871-2354, E-mail: fgoldste@alfred.edu

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Soils are often high in insoluble mineral and organic phosphates but deficient in available orthophosphate (Pi). Phosphorous is second only to nitrogen as an essential nutrient for plant growth and development. Soil amendment with fertilizer P, produced via chemical processing of rock phosphate ore, is an absolute requirement in order to feed the world's population. For over one hundred years workers have recognized the ability of soil microorganisms to solubilize Pi from insoluble (i.e. nutritionally unavailable) organic and mineral phosphates. Molecular characterization of the biochemical/genetic pathways of microbial P solubilization will enhance our understanding of phosphate cycling in natural ecosystems and make an essential contribution to the development of the renewable agricultural practices necessary for the long term environmental stability of our planet.

A wide range of microbial biosolubilization mechanisms exist, so that much of the global cycling of insoluble organic and inorganic soil phosphates is attributed to bacteria and fungi. The genetic and biochemical mechanisms for this solubilization are as varied as the spectrum of P-containing soil compounds.

High molecular weight organic phosphates are ubiquitous, whereas soil type and pH will control the chemistry of insoluble mineral phosphate precipitates. Large amounts of iron or aluminium phosphates are often found in acidic soils whereas, in arid to semiarid soils, calcium phosphates (CaPs) predominate. The limiting levels of Pi in most soils provides the ecophysiological basis for positing associations between plant roots and mineral phosphate solubilizing (MPS) and/or organic P solubilizing microorganisms. These associations are assumed to play an important role in phosphorus nutrition in many natural and agro-ecosystems. As a result, an enormous amount of research has been conducted involving isolation and characterization of MPS and organic P solubilizing microorganisms from a wide range of soils. In general, the goals have been to understand P cycling and/or to develop P biofertilizers analogous to biological nitrogen fixation. To date the results of these efforts have been problematic.

A general discussion of the full range of microbial mineral and organic phosphate biosolubilization is beyond the scope of this presentation, so the author will focus on calcium phosphates as a model (and widely applicable) system. Calcium phosphates may be dissolved by a wide array of microbe-generated organic acids. By comparison with organic phosphates, the molecular genetic and biochemical bases of bacterial transformations of poorly soluble CaPs has been difficult to resolve into a systematic field of study.

The reason may be the simplicity of the solubilization mechanism. Calcium phosphates are dissolved by acidification. Therefore, any bacterium that acidifies its external medium will show some level of MPS activity. In most soils, proton substitution reactions are driven by microbial production of organic acids; represented generically by the Equation 1:

Equation 1. [Ca²⁺]_m[PO₄^3-]_n + [HA] = [H⁺][PO₄ ^3- ] + [Ca²⁺][A^-]

There is no stoichiometry in Eq. 1 because of the complexity of CaP chemistry and the multiplicity of naturally occurring organic acids (HAs) with differing numbers of dissociable protons. It may be seen that any number of organic acids (HAs) may be substituted into Eq. 1 with varying efficacies depending on the solubility of the CaP, the number of acidic protons, and pKa(s) of the organic acid. Therefore, one is immediately confronted with the question of how to investigate the MPS phenomenon in a way that can determine if some bacteria produce acids as a strategy to make Pi bioavailable. In that case the organic acid production could be considered a phenotype, defined as a 'characteristic of the organism'. Alternatively, the proximity of organic acid-producing bacteria and CaPs within the soil will result in some solubilization. This could be called a MPS effect, as in 'to cause Pi to come into being'. As previously mentioned, an enormous amount of applied microbiology research has been conducted in this field.

It is generally accepted that the most common mechanism for the MPS phenomenon with respect to calcium phosphates is the acidification of the medium via biosynthesis and release of organic acids. However, the multiplicity of acid-generating metabolic pathways available to bacteria has made it impossible to develop a unified approach to the microbiology of CaP solubilization such as is available for the study of N₂ fixation; where all pathways must converge at some variation of the nitrogenase system. However, literally thousands of Gram-negative rhizobacterial isolates have been screened for the MPS trait on minimal CaP medium with glucose as the carbon source. When using these screening criteria, isolates expressing the direct oxidation pathway far surpass others in their ability to dissolve calcium phosphates. These bacteria have been designated as having a MPS⁺ phenotype. MPS⁺ bacteria can dissolve highly insoluble phosphates such as rock phosphate ore (RPO, flourapatite) because of the extremely low pKas of the glucose oxidation products; gluconic acid, and 2-ketogluconic acid (pKas of ~3.4 and ~2.6 respectively). In addition, since these acids are produced in the periplasmic space, protons are efficiently released into the extracellular medium, or rhizosphere space in vivo. Superior MPS⁺ strains not only have direct oxidation genes but express this metabolic pathway at a high level so that there is a direct correlation between acid production and CaP dissolution. It is also of interest to note that Pi starvation can induce the direct oxidation pathway in some strains. Therefore, a thorough review of the literature and twenty years of directed studies leads this author to the conclusion that, in many ecosystems, a relationship exists between highly efficacious Gram-negative MPS bacteria and the expression of the direct oxidation pathway. The question of rhizosphere mutualism and even symbiosis between efficacious MPS bacteria and higher plants remains to be clarified.

Direct oxidation (a.k.a. nonphosphorylating oxidation) is one of the four major metabolic pathways for glucose (aldose) utilization by bacteria. For many bacterial species, the direct oxidation pathway is the primary mechanism for aldose sugar utilization. The first oxidation is catalyzed by the quinoprotein glucose dehydrogenase; so named because it is a member of the group of bacterial enzymes that utilize the redox cofactor PQQ (2,7,9-tricarboxyl-1H-pyrrolo[2,3-f]quinoline-4,5-dione). PQQGDH is a membrane-bound dehydrogenase whose catalytic domain is located on the outer face of the cytoplasmic membrane. This enzyme transfers electrons from aldose sugars directly to ubiquinone in the plasmamembrane via two electron, two proton oxidations mediated by the cofactor PQQ. Direct oxidation of glucose to gluconic acid generates a transmembrane proton motive force (PMF) that may be used for bioenergetic and/or membrane transport functions while the dissociable proton of gluconic acid is available for CaP solubilization. MPS⁺ bacteria usually carry out the second periplasmic oxidation of gluconic acid to 2-ketogluconic acid via gluconate dehydrogenase.

We have proposed that the direct oxidation pathway is the metabolic basis for the superior MPS⁺ phenotype in Gram-negative bacteria. This provides workers with both a unifying metabolic strategy, and a set of biochemical and genetic probes with which to systematically identify and evaluate the role of a specific subpopulation of rhizosphere bacteria in P cycling. In addition, the demonstrated efficacy of the direct oxidation pathway for the dissolution of fluroapatites has provided a potential strategy for large-scale bioprocessing of rock phosphate ores. In terms of future research, successful molecular cloning of direct oxidation pathway genes provides the tools with which to study relationships between the population dynamics of MPS⁺ bacteria in the rhizosphere and Pi in the soil solution. Available probes include several apo-glucose dehydrogenase genes, PQQ biosynthesis genes, and at least one gluconate dehydrogenase gene. We have used such genetic probes to help demonstrate the presence of unique populations of Gram-negative MPS⁺ rhizobacteria in two alkaline desert soil environments where the levels of poorly soluble calcium phosphates are extremely high but Pi is undetectable in bulk soil extracts. Both MPS⁺ populations were capable of high levels of direct oxidation of glucose, and the presence of the quinoprotein glucose dehydrogenase was confirmed by both enzymatic and molecular biology assays. In one case, a unique rhizobacterial population of Enterobacter cloacae expressed the direct oxidation pathway only in the presence of compounds washed from the root of the host plant, Helianthus sp. providing preliminary evidence for mutualism in this highly alkaline soil (pH 10).

With respect to agriculture, bioprocessing of rock phosphate ore (RPO) to inorganic phosphate may provide an energy efficient, environmentally desirable alternative to current technology for industrial P fertilizer production. Bioconversion occurs at a low temperature and is more selective to phosphate extraction than conventional processes. This increased selectivity of attack may reduce the solubilization of undesirable ore contaminants such as radionuclides and toxic metals. The process uses carbohydrate as an energy/proton source as opposed to the 'wet-acid' chemical process that uses concentrated sulfuric acid. The bioprocessing of phosphate ore is not as sensitive to ore quality as are conventional approaches. This may allow lower grade ore deposits and tailings, not presently of any value, to be used. Finally, appropriate formulation may allow the bioprocess to be utilized for in situ bioconversion of RPO in the soil or even specifically in the rhizosphere of plant roots. Currently, we are implementing bioprocess engineering strategies that involve both conventional fermentation technology and advanced methods such as directed evolution to produce a system that can compete with the economics of conventional wet-acid phosphate facilities. In our approach, glucose produced via biomass conversion (an intermediate step in ethanol production) is used as feedstock for direct oxidation-mediated biosolubilization of rock phosphate ore. Looking towards the future it is reasonable to propose that, using the tools of biotechnology, biophosphorous fertilization is an achievable goal that lends itself well to the global imperative of sustainable agricultural production.