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Bioprocessing Of Rock

Phosphate Ore

The Development Of AProcess for Phosphoric acid[2000]Alan H. GoldsteinAlfred University, New York, USA

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Phosphorus participates in the reactions that keep plants alive, and is thus essential for all living organisms. Phosphorous is found in two different forms in soil: inorganic and organic.Inorganic phosphorusThe main inorganic forms of phosphorus in soil are H2PO4 - and HPO4 2-. This is the form in which phsophorus is used by plants. However, these ions can also adsorb onto the surface (or absorb into) solid matter in the soil. This phosphorus is then unavailable to plants.Organic phosphorusBetween 50 and 80% of phosphorus in soil is organic phosphorus. This comes from the breakdown of dead plants etc., as phosphorus is found in cell membranes and DNA in living organisms.

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Phosphate cycling in the soil

� Phosphorus is naturally available in the soil. However, there isn't usually enough available for plants to grow well. Phosphorus levels are reduced by crops raised and animals eating the plants then dying elsewhere so that the phosphorus is removed, and also by phosphorus being adsorbed into soil particles or washed away by excess rain. For this reason phosphate fertilizers are widely used.

� There are ways in which this influences phosphate cycling in the soil. Studies have concentrated on the reactions of added phosphate with soil constituents and on mechanisms controlling the amount of phosphate in solution.

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cycling in the soil…

� The inorganic reactions that control the concentration of phosphate ions in solution are:

� Precipitation-dissolution and � Sorption- desorption processes. � First reactions involve the formation and dissolving

of precipitates. The second reactions involve sorption and desorption of ions and molecules from the surfaces of mineral particles.

� The movement of phosphate into plants also influences soil solution concentrations and promotes dissolution and desorption reactions.

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INORGANIC PHOSPHORUS IN SOIL

� Although the phosphate ion can occur in three states of protonation, at pH values normally found in soils (4.5 - 6.2) H2PO4- and HPO4 2- are the dominant species.

� The role of biological immobilisation -decomposition is concerned with organic

phosphates.

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Introduction

� Rock phosphate ore (RPO = Ca10(PO4)6F2) may be leached by contacting the ore particles with a film of Gram negative mineral phosphate solubilizing bacteria (GnMPSb), under the appropriate growth conditions, giving the end product, phosphoric acid.

� In this “bioprocess”, the bacteria produce the strong organic acids that contact the ore particles and solubilize the phosphate.

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Genera of microbes

� Psudomonas

� Bacillus

� Penicilium

� Aspergillus

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Process involves much more than simple acid dissolution of the ore.

� In addition to acidification,in a contact bioreactor, with the bacterial biofilm on the surface of the ore particle, produce unique physicochemical conditions that result in true biocatalytic events that enhance the rate and efficacy of bioleaching of Pi from the ore.

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• Basic research on metabolic pathway has been completed by agricultural microbiologists. The bioprocess technology is currently (in 2000) in the development stage. •The remaining technical components required to bring a system online involve chemical and fermentation process engineering to optimize efficiency and yield parameters.

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Advantages of bioprocess

1. There is no phosphogypsum production. 2. There is apparently no soluble radioisotope

production 3. The process is ‘environmentally friendly’4. The bioprocess may become less expensive than

current technology 5. The bioprocessing of RPO uses carbohydrate as

an energy and proton source as opposed to the conventional wet process that uses sulfur, a nonrenewable resource.

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Advantages of bioprocess…

6. Finally, the bioprocessing of RPO is not as sensitive to ore quality as are conventional processes and the commercial application of this technology will allow lower grade ore bodies and tailings not presently of any value for processing to be used.

7.Therefore, the shift to a bioprocess-based technology will greatly increase the available phosphate ore reserves.

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Certain bacteria are highly efficient at dissolving calcium phosphates/ RPO. Goldstein’s laboratory identified the metabolic pathway of these bacteria to dissolve RPO and developed the use of these bacteria under lab. conditions, for the extraction of phosphoric acid from RPO.

Cited review articles:1) Goldstein, 1987, Molecular cloning andregulation of a mineral phosphate solubilizing gene from Erwinia herbicola. Bio/Technology. 5:72-74. 2) Goldstein et al, 1993, Separating Phosphate From Ores Via Bioprocessing. BIO/TECHNOLOGY, 11:1250-1254. 3) Goldstein, 1994, Solubilization Of Exogenous Phosphates By Gram Negative Bacteria. In, Cellular and Molecular Biology of Phosphate and Phosphorylated Compounds in Microorganisms. S. Silver et al, eds, ASM Washington, D.C pp. 197-203.

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Mineral Phosphate Solubilizing Phenotype

� Goldstein laboratory has shown that bacteria with a certain type of metabolism are superior to all other bacteria with respect to their ability to dissolve RPO.

� All highly efficacious Gram Negative- MPS Bacteria produce high levels of gluconic acid and/or 2-ketogluconic acid via the direct oxidation pathway.

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The Direct Oxidation Pathway:

� Gram Negative Bacteria And The Direct Oxidation Pathway: The outer perimeter of gram negative bacteria is composed of two membranes separated by a porous gel-like material and acid is produced here. The direct oxidation pathway also known as the ‘nonphosphorylating oxidation’pathway, involves the enzymatic conversion of glucose to gluconic acid and/or 2-ketogluconic acid on the outer face of the inner membrane.

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•Acid production actually occurs in the periplasmic space, where glucose, gluconic acid and 2-ketogluconic acid can enter in / out to the ore surface. and move freely. •Glucose dehydrogenase (GDH) is the enzyme that oxidizes glucose to gluconic acid (pKa ~3.6). This enzyme is anchored in the inner membrane but the catalytic surface is in the periplasmic space. Production of gluconic acid occurs functionally at the cell surface

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Gluconic acid dehydrogenase (a.k.a. gluconatedehydrogenase; GADH) is the second enzyme in the direct oxidation pathway. GADH converts gluconic acid to 2- ketogluconic acid (pKa ~ 2.4). Like glucose dehydrogenase, this enzyme is anchored in the inner membrane but the catalytic surface is in the periplasmic space. As a result, 2-ketogluconic acid will also freely diffuse out of the periplasmic space and make direct contact with the RPO.

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•Calcium phosphate compounds have a wide range of solubilities which, in general, follow an inverse relationship with the Ca/P ratios. •For example, monocalcium phosphate [Ca(H2PO4)2, Ca/P = 0.50] has a water solubility of 150,000 ppm at pH 7. • Whereas fluroapatite [Ca10(PO4)6F2, Ca/P=1.66] has a water solubility of 0.003 ppm.•Poorly soluble mineral phosphates such as fluroapatite or hydroxyapatite can only be effectively dissolved in aqueous solution under acidic conditions. This dissolution is the result of acid-mediated proton substitution for calcium.

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For fluroapatite and a generic acid HX that dissociates

to form H+ and X-:

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Besides the physicochemical reaction paths, there are 4 metabolic variables of state for the biodegradation system; there are, also, engineering variables of state for the bioreactor as well. These variables of state are not independent but, rather, form a dynamic interactive system that may be manipulated as a part of a total system design and engineering plan to optimize the yield of the bioprocess:

Molecular mechanism(s) of biodegradation of

RPO

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1. Biofilm Formation

It is highly probable that gram negative MPS bacteria grow as a biofilm on the surface of the ore particle. Biofilms create a unique growth environment in which microscopic domains or regions of space may exist where physicochemical conditions are extremely different from the average values measured in the bulk solution of the reactor tank.

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2. Microdomain Effects Within The Biofilm

� While the macroscopic pH of the bioreactor is often held at ~3.4, the effective proton activity (true acidity) at the surface of the ore particle may be much stronger.

� This is especially true if the bacteria is expressing both enzymes of the direct oxidation pathway so that a significant percentage of the gluconic acid (pKa~3.6) is immediately oxidized to 2-ketogluconic acid (pKa~2.4).

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Microdomain Effects Within The Biofilm-2

� The electrochemical potential of protons at the ore surface may be further modified by the unique surface electrochemistry of the biofilm and the polyanionic matrix thatforms the actual adhesion layer between the bacterium and the ore surface.

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3.Surface Electrochemistry & BiocatalysisAt The Bacteria/Ore Particle Interface

� the rate of dissolution of HAP is controlled by surface processes and depends more on the concentration of calcium than the concentration of phosphate as given by the function [Ca]x [PO4] where x may vary from 1.67 to 3.3 depending on solutionconditions and stoichiometry.

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4. Calcium chelation and/or electrostatic binding

� Under nonequlibrium conditions, even a small amount of Ca++ chelation/binding by free organic acids or the polyanionic matrix would be expected to dramatically enhance the rate of dissolution.

� Several studies have shown that sugar acids such as 2-ketogluconic acid have multiple conformations and, therefore, may chelate cations via unusual molecular mechanisms not predicted from equlibrium binding studies carried out in bulk solutions.

� In addition, polyanions can be highly effective at binding Ca++.

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Bioreactor design parameters

� Production of gluconic and 2-ketogluconic acids is an aerobic process so that essential bioreactor design parameters will need to include interactions between particle size, recycling and/or growth of microorganisms, rate of particle consumption, oxygen supply, substrate supply and removal of inhibitory end products.

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Bioreactor design strategies

� Reactor configuration for maintenance of maximum metabolic activity per unit cell and per unit volume to be evolved.

� Three phase system-- water, cell and solid substrate and air bubbles.

� High volume low value bioreactors must be designed to provide minimum cost and maximum effectiveness.

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Bioreactor design: Two Coupled 3 phase systems

� The efficient bioprocessing of RPO may involve coupling two ‘three-phase’ systems; the first involving the solid-phase fermentation of waste biomass to glucose.

� The second involving solid-phase bioleaching of the soluble phosphate from the ore matrix (the question of bioleaching vs. biodegradation depends to a large extent on the amount of matrix remaining after the Pi has been efficiently extracted).

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Bioreactor Configurations

� Solid phase bioprocessing occurs in slurry bioreactors, in which the solids are kept in suspension either by mechanical agitation, aeration or a combination of both. Specific designs include airlift fermenters (Bos et al,1988), aerated troughs (Andrews, 1990), various modifications of the fluidized-bed (e.g. Asif et. al., 1993), and various modifications of the slurry agitator (e.g. Griffin et. al., 1990). The contact bioreactor developed at INEL and described by Goldstein et al (1993) may be considered to be a microscale version of a modified slurry agitator.

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Biofilm microscopy indications:

� Application of confocal scanning laser microscopy clearly shows that biofilm bacteria grow in matrix-enclosed microcolonies interspersed with less dense regions of the matrix that include highly permeable water channels.

� The water channels that anastomose throughout microbial biofilms provide direct high-permeability access from the bulk fluid to the colonized surface. It has further been shown that convective flow patterns are operative within the water channels.

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ultrastructure of the biofilm:

� From the standpoint of the physicochemical variables discussed above, the ultrastructure of the biofilm provides a system almost perfectly designed for the biodegradation process.

� Assuming extreme environmental conditions at the ore particle surface produce the solubilizationproducts, these products will be carried into the bulk phase of the solution by convective currents within the biofilm.

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poly-anionic matrix…

� The polyanionic matrix in which the biofilm cells are embedded could play a direct role in the surface electrochemistry of the dissolution process. Christofferson and Christofferson (1982) have calculated the electrochemical activation energy for transport of Ca++ ions from the HAP surface to the solution (therate-limiting step in HAP dissolution).

� According to this work, adherence of a polyanionic material would favorably affect the energetics of solvation of the Ca++ ions by lowering the electrical potential and therefore the freeenergy for Ca++ in thesolution (actually interphase space) directly adjacent to the mineral surface.

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Chelation….

� Once in solution, any chelation by gluconate, 2-ketogluconate and/or Ca++ binding by the polyanionic matrix would effectively decrease the activity coefficient of the solvated Ca++

� Likewise, microdomains of high acidity would drive the dissolution reaction since (according to these same authors) hydrogen ions catalyse the exchange of phosphates between the crystal surface and the solution.

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The possibility of Ca++ chelation within microdomains of the biofilm cannot be ignored. Van Bekkum and coworkers (c.f. van Duin, 1989) have used oxygen-17 NMR shifts to develop a general coordination-ionization scheme for polyhydroxy carboxylic acids such as gluconic acid and 2-ketogluconic acid. Their data clearly show that both gluconic and 2-ketogluconic acid are capable of Ca++ coordination at low pH. This is especially true for gluconic acid where, under acidic conditions, bidentate coordination of the cation occurs via interaction with the hydroxy and carboxylic acid moieties.

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Biocatalytic event…

� Taken in combination, these two affects are equivalent to a truecatalytic event so that the enhanced efficiency of solubilizationseen in the contact bioreactor may, in part, result from the bacteria and their associated biofilm matrix acting as biocatalystic system.

� At the present time, the effective pH or partial pressure of O2 at the ore particle surface is not known. Fluorescent probes and microelectrodes have been used to measure these parameters in model biofilm systems including Pseudomonas aeruginosa (a close relative of Pseudomonas cepacia now classified as Burkholderia cepacia; the bacterium currently used in the contact bioreactor).

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Continued…

� These data show that biofilms are generally aerobic which, in turn, should allow the direct oxidation of glucose to proceed. In the microcolony itself, both the concentration of acid and the pKa will be a function of interphase conditions which may be viewed as a continuum that moves from the periplasm through the extracellularpolyanionic matrix to the surface of the ore particle.

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formation of biofilms…

� Adhesion to a surface and/or the formation of biofilms is now known to trigger changes in bacterial metabolism including changes in gene expression. The degree to which the direct oxidation pathway is expressed and, consequently, the extent to which gluconic acid and/or 2-ketogluconic acid is produced in the ‘dissimilatory bypass’ mode is highly variable.

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triggered changes in bacterial metabolism?

� In the bioreactor, this level may be much higher in the biofilm bacteria than the bacteria growing in the planktonic population within the bulk fluid of the stirred tank bioreactor. As a result, the average level of gluconic acid production (as measured by % conversion of glucose in the feedstock) may not represent the effective concentration of acid at the ore surface.

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Engg. Analysis needed

� Roles for some of the candidate variables of state identified in this section may be validated within the variation of parameters format that would be expected as part of an optimized implementation of the bioprocess and metabolic engineering systems.

� Therefore, design of a pilot-scale bioreactor to test the commercial feasibility of the bioprocess must include strong chemical engineering and fermentation engineering components.

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Selected References1:

� Anderson, S., et al 1985. Production of 2-keto-L-gluconate, an intermediate in L-ascorbate synthesis, by a genetically modified Erwinia herbicola . Science 230:144-149.

� Andrews, G.F. 1990. Large-scale bioprocessing of solids. Biotechnol. Prog. 6: 225-229.

� Andrews, G.F. et al 1993. Heaps as bioreactors. Applied Biochemistry and Biotechnology39/40: 427-433

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Selected References2:

� Bos, P., et al 1988. Feasibility of a Dutch process for microbial desulfurization of coal. Resour.Conserv. Recycl. 1:279.

� Christofferson, J. and Christofferson, M.R. 1979. Kinetics of dissolution of calcium hydroxyapatite II. Journal of Crystal Growth 47:671-679.

� Christofferson, J. and Christofferson, M.R. 1982. Kinetics of dissolution of calcium hydroxyapatite V. Journal of Crystal Growth 57:21-26.

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Selected References3:

� Goldstein, A.H. 1986. Bacterial mineral phosphate. Am. J. Alt. Agric. 1(2):51-57.

� Goldstein, A.H. and S.T. Liu. 1987. Molecular cloning and regulation of a mineral phosphatesolubilizing gene from Erwinia herbicola. BIO/TECHNOLOGY. 5:72-74.

� Goldstein, A.H., R.D. Rogers and G. Mead. 1993. Separating Phosphate From Ores ViaBioprocessing. BIO/TECHNOLOGY, 11:1250-1254.

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Selected References4:

� Goldstein, A.H. 1994. Solubilization Of Exogenous Phosphates By Gram Negative Bacteria. In, Cellular and Molecular Biology ofPhosphate and Phosphorylated Compounds in Microorganisms. S. Silver et al, eds, ASM Washington, D.C pp. 197-203.

� Goldstein, A.H., 1995. Recent progress in understanding the molecular genetics and biochemistry of calcium phosphate solubilization by gram negative bacteria. Biological Agriculture & Horticulture, 12:185-193.

� Griffin, E.A., et al 1990. Bioreactor development with respect to process constraints inposed by bio-oxidation and waste remediation. Applied Biochemistry and Biotechnology 24/25: 627-635

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Selected References5:

� Biochim Biophys Acta. 2003 Apr 11;1647(1-2):266-71.Research on the metabolic engineering of the direct oxidation pathway for extraction of phosphate from ore has generated preliminary evidence for PQQ biosynthesis in Escherichia coli as well as a possible role for the highly conserved region of quinoprotein dehydrogenases.

Goldstein A, Lester T, Brown J.

Biomedical Materials Engineering Science Program, NYSCC at Alfred University, 2 Pine Street, Alfred, NY 14802, USA. fgoldste@alfred,edu

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Appendix

Miscellaneous papers

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IFA Technical Conference, New Orleans, U.S.A., 1-4, October 2000

� Bio-processing Of Rock Phosphate Ore: Essential Technical Considerations For The Development Of A Successful Commercial Technology.,

� Alan H. Goldstein� Alfred University, New York, USA� Fierer Chair of Molecular Cell Biology, and Graduate

Program in Biomedical Materials Engineering Science, School of Ceramic Engineering and Materials Science.

� E-mail : fgoldste@alfred.edu

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FEMS Microbiology Ecology

Volume 30 Page 295 - December 1999

doi:10.1111/j.1574-6941.1999.tb00657.x Volume 30 Issue 4

� Evidence for mutualism between a plant growing in a phosphate-limited desert environment and a mineral phosphate solubilizing (MPS) rhizobacteriumAlan H. Goldsteina,b,*, Kate Bravermana, Nelson Osoriob

� Abstract: Alkaline desert soils are high in insoluble calcium phosphates but deficient in soluble orthophosphate (Pi) essential for plant growth. In this extreme environment, one adaptive strategy could involve specific associations between plant roots and mineral phosphate solubilizing (MPS) bacteria. The most efficient MPS phenotype in Gram-negative bacteria results from extracellular oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase.

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FEMS Microbiology Ecology……

� A unique bacterial population isolated from the roots of Helianthus annus jaegeri growing at the edge of an alkaline dry lake in the Mojave Desert showed no MPS activity and no gluconic acid production. Addition of a concentrated solution containing material washed from the roots to these bacteria in culture resulted in production of high levels of gluconic acid.

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FEMS Microbiology Ecology……

� This effect was mimicked by addition of the essential glucose dehydrogenase redoxcofactor 2,7,9-tricarboxyl-1H-pyrrolo[2,3]-quinoline-4,5-dione (PQQ) but the bioactive component was not PQQ. DNA hybridization data confirmed that this soil bacterium carried a gene with homology to the Escherichia coliquinoprotein glucose dehydrogenase.

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FEMS Microbiology Ecology……

� These data suggest that expression of the direct oxidation pathway in this bacterium may be regulated by signaling between the bacteria and the plant root. The resultant acidification of the rhizosphere may play a role in nutrient availability and/or other ecophysiological parameters essential for the survival of this desert plant.

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Research on the metabolic engineering of the direct

oxidation pathway for extraction of phosphate from ore

� It has generated preliminary evidence for PQQ biosynthesis in Escherichia coli as well as a possible role for the highly conserved region of quinoprotein dehydrogenases.

Goldstein A, Lester T, Brown J.

Biomedical Materials Engineering Science Program, NYSCC at Alfred University, 2 Pine Street, Alfred, NY 14802, USA. fgoldste@alfred,edu

The ability of some bacteria to dissolve poorly soluble calcium phosphates (CaPs) has been termed 'mineral phosphate solubilizing' (MPS). Since most microorganisms and plants must assimilate P via membrane transport, biotransformation of CaP into soluble phosphate is considered an essential component of the global P cycle. In many Gram-negative bacteria, strong organic acids produced in the periplasm via the direct oxidation pathway have been shown to dissolve CaP in the adjacent environment. Therefore, the quinoprotein glucose dehydrogenase (PQQGDH) may function in the ecophysiology of many soil bacteria.

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Research on the metabolic engineering of the direct

oxidation pathway….

� Interest exists in using MPS bacteria for industrial bioprocessing of rock phosphate ore (a substituted fluroapatite) or even for direct inoculation of soils as a‘ biofertilizer ' analogous to nitrogen fixation. Superior MPS bacteria were studied for 20 years . Screening genomic libraries in the appropriate E. coli genetic background can 'trap' PQQ or GDH genes from these bacteria via functional complementation. In setting the 'trap' for PQQ genes, we have identified DNA fragments that apparently induce PQQGDH activity in E. coli with no sequence homology to known PQQ genes. These data suggest that E. coli may have an alternative, inducible PQQ biosynthesis pathway. Finally, a novel protein engineering strategy to increase the catalytic rate of PQQGDH has emerged and will be discussed.