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Phosphate Ore
The Development Of A
Process for Phosphoric acid
[2000]
Alan H. Goldstein
Alfred University, New York, USA
1
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 phosphorus
The 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 phosphorus
Between 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.
2
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.
3
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.
4
INORGANIC PHOSPHORUS IN SOIL
5
Introduction
6
Genera of microbes
Psudomonas
Bacillus
Penicilium
Aspergillus
7
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.
8
• 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.
9
10
Advantages of bioprocess
11
Advantages of bioprocess…
12
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 and
regulation 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.
13
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.
14
The Direct Oxidation Pathway:
15
•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
16
Gluconic acid dehydrogenase (a.k.a. gluconate
dehydrogenase; 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.
17
•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.
18
For fluroapatite and a generic acid HX that dissociates
to form H+ and X-:
19
Molecular mechanism(s) of biodegradation
of
RPO
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:
20
1. Biofilm Formation
21
2. Microdomain Effects Within The Biofilm
22
Microdomain Effects Within
The Biofilm-2
23
3.Surface Electrochemistry & Biocatalysis
At The Bacteria/Ore Particle Interface
24
4. Calcium chelation and/or electrostatic binding
26
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.
27
Bioreactor design: Two Coupled 3 phase systems
28
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.
29
Biofilm microscopy indications:
30
ultrastructure of the biofilm:
31
poly-anionic matrix…
32
Chelation….
33
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.
34
Biocatalytic event…
35
Continued…
36
formation of biofilms…
37
triggered changes in bacterial metabolism?
38
Engg. Analysis needed
39
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 Biotechnology
39/40: 427-433
40
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.
41
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 phosphate
solubilizing gene from Erwinia herbicola.
BIO/TECHNOLOGY. 5:72-74.
Goldstein, A.H., R.D. Rogers and G. Mead. 1993.
Separating Phosphate From Ores Via
Bioprocessing. BIO/TECHNOLOGY, 11:1250-1254.
42
Selected References4:
43
Selected References5:
44
Appendix
Miscellaneous papers
45
IFA Technical Conference, New Orleans, U.S.A., 1-4,
October 2000
46
FEMS Microbiology Ecology
Volume 30 Page 295 - December 1999
doi:10.1111/j.1574-6941.1999.tb00657.x Volume 30 Issue 4
47
FEMS Microbiology Ecology……
48
FEMS Microbiology Ecology……
49
FEMS Microbiology Ecology……
50
Research on the metabolic engineering of the direct
oxidation pathway for extraction of phosphate from ore
51
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.
52