CA1207258A - Stimulation of bacterial growth by inorganic pyrophosphate - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A process for the growth of microorganisms wherein inorganic pyrophosphate is used as an energy source to generate adenosine triphosphate is disclosed. Microorganisms grow on a medium containing a fixed carbon source supplemented with inorganic pyrophosphate. This process can be used to overcome the problem of low growth or slow growth microorganisms used in commercial or industrial processes such as leaching of low grade pyrite ores, desulfurization of coal, conversion of biomass or cellulose to methanol, and conversion of biomass or cellulose to ethanol.

Description

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STIMULATION OE BACTERIAL GROWTH
BY INORGANIC PYROPHOSPHATE

Technical Field This invention relates to the growth of microorganisms using inorganic pyrophosphate as an energy source. This invention overcomes low or slow growth problems in many species of microorganisms,~ar~icularly species used in important commercial and industrial processes.

Background Art ,10 Inorganic pyrophosphate (PPi) has been proposed (F. Lipmann, The Origins of Prebiological Systems, pp. 261-271 Mir., Moscow, 1969) as an evolutionary precursor of adenosine triphosphate (ATP), and more recently the compound has been demonstrated to be involved in a number of energy yielding reactions (R. E.
Reeves, TIBS 1:53, 1976; H. G. Wood, W. E. O'Brien, G. Michaels, Adv. Enzymol., 45:85, 1977; K. S. Lam, C. B. Kasper, Proc. Natl.
Acad. Sci., 77t1927, 1980; N. W. Carnal, C. C. Black, Biochem.
Biophys. Res. Commun., 86:20, 1979; D. C. Sabularse and R~ L. Anderson, Biochem. Biophys. Res. Commun., 100:1423, 1981).
U.S. Patent No. 3,960,664 and U.S. Patent No. 3,010,876 disclose inorganic pyrophosphate as a minor component of growth media; however, U.S. Patent No. 3,960~664 and U.S.
Patent No. 3,010,876 do not mention the use of inorganic pyrophosphate as an energy source.

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Disclosure of_Invention The bioenergetics of respiratory sulfate reduction by two of the described genera of sulfate reducing bacteria, Desulfovibrio and Desulfotomaculum, are fundamentally different _. __ _ (C.L. Liu, H. D. Peck, Jr., J. Bacteriol., 145:966, 1981). In the case of Desulfovibrio, the inorganic pyrophosphate (PPi), produced from adenosine triphosphate ~ATP) by the enzyme adenosine triphosphate sulfurylase (Eq. 1) in the first enzymatic step of Eq. 1 ATP + SO4 ATP + PPi respiratory sulfate reduction, is hydrolyzed to orthophosphate (Pi) by inorganic pyrophosphatase (Eq. 2).

Eq. 2 PPi + H20 --~2Pi Thus, the chemical energy in the anhydride bond of inorganic pyrophospha~e (PPi) is not conserved and, in order to obtain a new yield oP adenosine triphosphate (ATP) during growth on a la~tate-sulPate médium, Desul~ovibrio species carry out electron transPer coupled phosphorylation in this growth mode.
In contrast, Desulfotomaculum species are able to conserve the bond energy of the pyrophosphate produced by adenosine triphosphate ~ATP) sulfurylase ~Eq. 1) by means of the enzyme, inorganic pyrophosphate acetate phosphotransferase (Eq. 3) (R. E. Reeves, J. B. Guthrie, Biochem. Biophys. Res. Commun., 66:1389, 1975).

Eq. 3 Acetate + PPi Acetyl phosphate + Pi Adenosine triphosphate (ATP) can then be produced from acetyl phosphate and adenosine diphosphate (ADP) by acetate kinase (Eq. 4).

Eq. 4 ADP + acetyl phosphate acetate + ATP

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These two enzymatic reactions allow Desulfotomaculum species to generate one high energy phosphate by substrate level phosphorylation per sulfate reduced to sulfide during growth with lactate on the lactate-sulfate med;um.
It is not necessary for Desulfotomaculum species to carry out electron transfer coupled phosphorylation during growth with lactate and sulfate.
Utilization of inorganic pyrophosphate as an energy source for the growth of microorganisms is an entirely new observation and a unique concept regarding the energy metabolism of anaerobic bacteria and some aerobic microorganisms. It appears to have many important ramifications for basic biology, microbial ecology, and applied microbiology. In the sulfate-reducing species belonging to the genus Desulfotomaculum, utilizat-ion of inorganic pyrophosphate as an energy source represents overall the simplest adenosine triphosphate generating system in the biological world requiring only one specific enzyme, phyrophosphate acetate phosphotrans-ferase, plus the ubiquitous, acetate kinase.
From the standpoint of microbial ecology, the widespread occurrence of inorganic pyrophosphate utilization suggests that inorganic pyro-phosphate functions as a new type of energy transfer system. The demonstrat-ion of inorganic pyrophosphats as an important part of the phosphorus cycle will represent a major contribution Jo the understanding of microorganisms and their relationships in different ecosystems such as the salt water marsh and sediments, fresh water marsh and sediments, anaerobic sludge digestors, and the rumen. The stimulation of microbial growth by inorganic pyrophosphate has a number of potential applications in applied micro-biology, and inorganic pyrophosphate is a common and inexpensive chemical.
Accordingly, the present invention seeks to provide a process for the growth of microorganisms wherein inorganic pyrophosphate is used as an energy source.
In one broad aspect, the invention pertains to a process for cul-tivating a microorganism~which comprises cultivating a microorganism in a growth medium containing an inorganic pyrophosphate, wherein the micro-organism is capable of utilizing the inorganic pyrophosphate as an energy source for internal generation of adenosine triphosphate when the in-organic pyrophosphate is externally suppied to the microorganism.
The invention pertains further to a process for the stimulation of the growth rate of a microorganism which comprises adding an inorganic pyro-phosphate to a composition which comprises a microorganism in a growth medium which does not contain the inorganic pyrophosphate wherein the 25i5~

microorganism is capable of utilizing the inorganic pyrophosphate as an energy source for internal generation of adenosine triphosphate when the inorganic pyrophosphate is externally supplied to the microorganism.
The invention further seeks to provide a process for using in-organic pyrophosphate as an energy source to stimulate growth rates ofmicroorganisms used in commercial and industrial applications such as leaching of low grade pyrite ores, desulfurization of coal, conversion of biomass to ethanol, and conversion of biomass to methane.
These and other aspects and advantages of this invention will become apparent from a consideration of the accompanying specification and claims.
Brief Description of the Drawings Figure 1 shows the effect of inorganic pyrophosphate concentration on the growth of Desulfotomaculum ruminis. Growth conditions were the same as in Example II, below, using the basal medium and with varying concentrations of inorganic pyrophosphate. Figure 2 shows two photo-micrographs of microorganisms growing in inorganic pyrop~osphate (PPi)enrichments of marine mud using the basal medium supplemented with 2.5%
sodium chloride as described in Example It, below.
Modes for Carryin out the Invention g _esulfotomaculum nigrificans, Desulfotomaculum ruminis, and Desulfoto-maculum orientis were grown on a medium containing inorganic pyrophosphate,acetate, yeast extract, sulfate, and salts. In the sulfate reducing Desulfotomaculum species of microorganisms, the use of inorganic pyrophosphate as an energy source in a process for growth requires only one specific enzyme, pyrophosphate acetate phosphotransferase. The ubiquitous enzyme, acetate kinase, is also required. Adenosine triphosphate is generated by this system. Also, crude enrichment cultures from a marine spartina marsh and fresh water marshes were similarly grown using inorganic pyrophosphate as an energy source.
Example I, below, describes conditions for the anaerobic growth of Desulfotomaculum nigrificans on inorganic pyrophosphate.
Table 1, below, compares growth on various media. The basal medium, containing acetate, yeast extract, sulfate, and salts does not I' ~LZ~
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support growth of the microorganism. When the basal medium was supplemented with inorganic pyrophosphate, growth was better than obtained under usual growth conditions with lactate plus sulfate. On the basal medium, inorganic pyrophosphate does not stimulate the growth of Desulfovibrio vulgaris; and orthophosphate9 equivlent to the added inorganic pyrophosphate, does not support the growth of Desulfotomaculum nigrificans, Desulfotomaculum ruminis, and Desulfotomaculum orientis. For optimal growth of Desulfotomaculu_ nigrificans on inorganic pyrophosphate, acetate, yeast extract, and sulfate were required; acetate and yeast extract were the fixed carbon source3 and sulfate provided the microorganisms with an electron sink with which to adjust the oxidation level of the fixed carbon source in the basal medium. The con-centration of sulfate was only 1/10 of that used in the usuallactate-sulfate medium, but the requirement for acetate was unexpectedly high with little growth occurring below a concentration of 0.2%. The physiological basis for this high acetate requirement may have involved the bioenergetics oE the permeation of acetate into Desulfotomaculu _igrificans.
The stimulatory effect of inorganic pyrophosphate on growth does not appear to be due to the facilitation of anaerobic acetate oxidation by inorganic pyrophosphate, as the ratio of acetate disappearance to sulfide production was 3:14 rather than the expected ration of 1:1. Similar growth responses were found for Desulfotomaculum ruminis and _esulfotomaculum orientis; thereforej inorganic pyrophosphate served as an energy source for growth of these anaerobic sulfate reducing microorganism.

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Table 1. Requirements for the Growth of Desulfotomaculum Utilizing Inorganic Pyrophosphate (PPi) as a Source of Energy Additions and/or Deletions Growth* (O.D.) Basal Medium 0.019 Basal Medium plus PPi 0.628 Basal Medium minus Sulfate plus PPi 0.019 Basal Medium minus Acetate plus PPi 0~095 Basal Medium minus Yeast Extract plus PPi 0.042 nasal Medium minus Acetate and Yeast extract plus PPi 0.036 Lactate-Sulfate Medium 0.505 *Measured at 580 nm; average of duplicate flasks after forty-eight hours incubation at 55C under argon.

The data for Example II, below, illustrated in Figure 1 shows the growth response of Desulfoto aculum ruminis to increasing amounts of inorganic pyrophosphate. The growth response was proportional to inorganic pyrophosphate concentra-tions up to 0.04% and growth was accompanied by the hydrolysis of inorganic pyrophosphate. In the absence of growth, there was little hydrolysis of added inorganic pyrophosphate. Above 0.05%, inorganic pyrophosphate growth was inhibited which may have been due to alkalization of the medium (pi 8.5) as a result of inorganic pyrophosphate hydrolysis. Similar results were obtained with Desulfotomaculum nigrificans and Desulfoto-_ culum orientis.

The enzymatic complement of cells of Desulfotomaculum orienti_ grown on lactate-sulfate media were compared with that of cells grown on basal medium plus inorganic pyrophosphate.
Growth media is described in Example III, below. The specific activities of various enzymes found in inorganic pyrophosphate and lactate-sulfate grown cells of Desulfotomaculu_ orientis are shown in Table 2, below. The reductases of respiratory sulfate reduction, APS reductase, thiosulfate reductase, bisulfite reductase, and adenosine triphosphate sulfurylase had about the same levels of activity in each cell preparation.

7~ ~5 Fumarate reductase was absent in both inorganic pyrophosphate grown and lactate-sulfate grown cells, and nitrite reductase, formate dehydrogenase, inorganic pyrophosphate acetate kinase, pyrophosphatase and pyruvate dehydrogenase were present at similar specific activities. The reason for the significantly higher hydrogenase in inorganic pyrophosphate grown cells may have been due to difficulties with the assay procedure.
The unique occurrence of these enzymes was also confirmed for _ sulfotomaculum nigrificans and Desulfotomaculum ruminis which_ _ _ _ _ _ indicated that the cells grown on inorganic pyrophosphate exhibit no basic changes in their metabolic pattern.

Enzymatic activities were determined by the following standard assay procedures (Odom, J. M. and Peck? H. D. Jr., J. Bacteriol. 147:161-169, 1981. Enzyme assays. Benzyl viologen- or methyl viologen-coupled nitrite, sulfite, fumarate, and ~hiosulfate reductases and hydrogenase were all assayed manometrically. Reaction mixtures consisted of 100 mM (pal 7.4) phosphate, 5.0 mM benzyl or methyl viologen, and 20.0 mM
sùlfite, fumarate, or thiosulfate or 5.0 mM nitrite in a total volume of 1.0 ml. Partially purified hydrogenase (through the first DEAE column) from D vulgaris (van der Westen, H., S. G.
Mayhew, and C. Veeger, FEBS Lett. 86:1Z2-126, 1978) was added in all manometric viologen-linked assays except the hydrogenase assay. Pyruvate-BV2+ and formate-BV reductases were determined spectrophotometrically at 545 nm in a Beckman model 25 spectrophotometer. Reaction mixtures contained 5.0 mM BV
lO.O mM dithiothreitol, 100 mM potassium phosphate buffer (pH 7.4), and 20 mM sodium formate for the formate BV2 reaction and 5.0 mM BV 2.0 mM reduced coenzyme A, 100 mM potassium phosphate (pH 7.4), and 20.0 mM sodium pyruvate for the pyruvate-BV reaction. Both assays were performed under argon in Thunberg covettes. Succinate ferricyanide reductase and APS reductase assays were performed with a spectrophotometer, under air, at 420 nm, using 40.0 mM adenosine monophosphate 30.0 mM sulfite-l.O mM ferricyanide-100 mM potassium phosphate, pH 7.4, for the APS reductase reaction (Bramlett, R. and H. D.
Peck, Jr., J. Biol. Chem. 250:2979-2986, 1975) and 20.0 mM
potassium succinate-lO mM ferricyanide-100 mM potassium phosphate, '`~

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pH 7.4, for the succinate-f~rricyanide reductase reaction.
C-type cytochromes were determined by difference spectroscopy at 538 to 551 nm (40). For membrane fractions and whole cells, difference spectra of 02-oxidized and dithionate-reduced membranes were measured at liquid nitrogen temperatures in an Aminco DW-2 spectrophotometer with a l-mm light path.
Changes in the apparent extinction coefficients at liquid nitrogen temperatures were taken into account by measuring the difference spectra of known amounts of cytochrome c3 (Mr=
1~,000) at room temperature (Shipp, W. S., Arch. Biochem.
Biophys. 150:459-472, 1972~. Cytochrome b was measured by differences in the absorption spectrum of its pyridine hemochromogene. Flavin adenine dinucleotide and flavin mononucleotide were determined by fluorescence at acid and neutral pH (Siegal, L. M., Methods Enzymol. 53D:419-429, 1979).
Menaquinone was determined by the method of Kroger (Kroger, A., Methods Enæymol. 53D:579-591, 1979), and non-heme iron was assayed by measuring color formation with o-phenanthroline ~Beinert, H., Anal. Biochem. 20:325-334, 1967). Protein was determined by the biuret method Cornell A. G.l G. J.
Bardawill, and M. M. David, J. Biol. Chem. 177:751-766, 1949).
Liu, Chi-Li and Peck, H. D. Jr., J. Bacteriol. 145:966-973, 1981~. Assays. Adenosine triphosphate sulfurylase was determined with MoO4 as described by Wilson and Bandurski ; 25 (Wilson, L. G. and R. S. Bandurski, J. Biol. Chem. 233:975-9~1, 1958), and inorganic pyrophosphatase was determined by the method of Akagi and Campbell (Akagi, J. M. and L. L. Campbell, J. Bacteriol. 84:1194-1201, 1962). Inorganic pyrophosphate acetate phosphotransferase was assayed by using the conditions of Reeves and Guthrie (Reeves, R. E. and J. B. Guthrie, Biochem. Biophys. Res. Commun. 66:1389-1395, 1975), except that acetyl phosphate (Lipmann, F. and L. C. Tuttle, J. Biol.
Chem. 159:21-28, 1945) produced from acetate plus pyrophosphate was determined. Sulfide was measured by the method of Siegel Siegel, L. M., Anal. Biochem. 11:126-32, 1965), and protein was measured by the biuret method (Levin, R. and R. W. Braver J. Lab. Clin. Med. 38:474-479, 1951). Lactate and acetate were measured with a Varian Aerograph 2700 gas chromatograph equipped with a hydrogen flame ionization detector.

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g Table 2. enzymatic Activities in Extracts oE Desulfotomaculum orientis Grown on Basal Medium plus Inorganic Pyrophosphate (PPi) and Lactate-Sulfate Medium enzymatic Activities Enzymes Specific Activity (n mole/min/mg) Lactate-Sulfate Basal Medium Medium plus PP
Bisulfite Reductase 64.3 57.1 Nitrite Reductase119.6 153.8 Thiosulfate Reductase 23.7 21.5 Fumarate Reductase 0 0 APS Reductase 397 385 Formate Dehydrogenase 64 87.1 Hydrogenase 16.2 85.2 Pyruvate Dehydrogenase 107 139 ~denosine Triphosphate Sulfurylase 140 151 Pyrophosphate Acetate Phosphotransferase Inorganic Phyrophosphatase There is no a priori reason to believe that the utilization of inorganic pyrophosphate as an energy source for growth was limited to the genus Desulfotomaculum. ,The growth of microorganisms of other genera on basal medium plus inorganic pyrophosphate was tested using enrichment cultures of micro-organisms. Specifically, microorganisms from mud samples obtained from a salt water spartina marsh were utilized to inoculate the basal medium supplemented with sodium chloride with and without inorganic pyrophosphate under anaerobic conditions as described in Example IV, below. Within twenty-four hours at 37C the anaerobic medium containing inorganic pyrophosphate showed extensive microbial growth and photo-micrographs of the mi'cr~rganisms in such enrichments weremade, The photomicrographs of these enrichment cultures con-tained a surprising high and unexpected number of morphological types of microorganisms. Similar results were obtained with inorganic pyrophosphate enrichment grown cultures from fresh water environments utilizing the same medium minus sodium chloride as shown in Example V, below. The diversity of cell-types showed that the use of inorganic pyrophosphate as anenergy source is not restricted to the genera Desulfotomaculum which is characterized by spore-forming rods. Some initial isolates from these enrichment cultures were non-sulfate reducing microorganisms and did not require acetate or sulfate for growth.

Desulfotomaculum species, Methanobacteriu_ species, and _ thanosarcina species are directly involved in the microbial community responsible for the conversion of cellulose, including biomasss and organic wastes, to methane and carbon dioxide, Desulfotomaculum species convert fermentation products such as lactate fatty acids and alcohols to acetate, carbon clioxidc and hydrogen; Methanobacterium species conv&rt carbon dioxide and hydrogen to m&thane; and Methanosarcln_ species convert acetate to methane and carbon dioxide. The conversion of fermentation products and production of methane from acetate or hydrogen plus C02 both, as described above, are limiting steps in this important biological process and the growth or physiological processes characteristic of these bacteria are stimulated by inorganic pyrophosphate as described in Example IV, below. The stimulation of methane formation by inorganic pyrophosphate with crude cellulose enrichments from a fresh water marsh has been demonstrated as shown in Example VII, below.

Methanosarcina barkerii has been shown directly to produce methane at an increased rate from acetate in the presence of inorganic pyrophosphate as shown in Example VIII, below.

The growth of Thermoanaerobacter ethanolic_s, a thermophilic fermen~ative anaerobe, on inorganic pyrophosphate was unexpected but the fact that these types of anaerobic organisms can metabolize inorganic pyrophosphate suggests a process wherein inorganic pyrophosphate is used to modify or alter the pattern of fermentation products. Since Thermoanaerobacter ethanolicus forms from 1.0 moles of glucose, 1.8 moles of ethanol, 0.1 moles of acetate, and 1.0 moles lactate, the accumulation of acetate and lactate limits the usefulness of Thermoanaerobacter _ hanolicus for the continuous production of ethanol.__ Thiobaeillus species are microorganisms used commercially for the leaching of low grade pyrite ores which is a slow process, due largely to the growth rates of these microorganisms.
The addition of inorganic pyrophosphate accelerates the leaching process by increasing the initial growth rates of these m;eroorganisms as shown in Example X, below. A reduced residenee time increases the eapacity of existing faeilities used in this process.

A second aspect of microbial leaching by Thiobaeillus speeies additions of inorganie pyrophosphate aeeelerate the desulfurization process by inereasing the initial growth lS rates of these mieroorganisms thereby providing the inereased baeterial mass required for the proeess. A reduced residenee time or the coal slurry makes the proeess eeonomieally viable.

Mierobial proeesses are utilized for the industrial produetion of a large number oE enzymes, fine biochemieals, and pharmaeeutieals. Addition of inorganie pyrophosphate to produetion media inereases the yield of these products and mier0rganism. For examples, the yield of vitamin B12 by Methanosarcina barkerii is increased by the addition of in-organic pyrophosphate as shown in Example XII, below.

The inorganie pyrophosphate acetate phosphotransferase represents the simplest biological system for produeing ATP
from a substrate. Using "state of the art" teehniques for the transfer of deoxyribonueleie aeid (DNA) from one microorganism to another, U.S. Patent No. 4,237,224 (Cohen and Boyer, Proeess for Produeing Biologieally Funetional Molecular Chimeras is a process which is known in the art. Method and compositions are provided for replication and expression of exogenous genes F~

~Z~7;~S8 in microorganisms. Plasmids or virus deoxyribonucleic acid are cleaved to provide a biologically functional replicon with a desired phenotypical property. The replicon is inserted into a microorganism cell by transformation. Isolation of the transformants provides cells for rep:Lication and expression of the deoxyribonucleic acid molecules present in the modified plasmid. The method provides a convenient and efficient way to introduce genetic capability into microorganisms for the production of nucleic acids and proteins, such as medically or commercially useful enzymes, which may have direct usefulness, or may find expression in the production of drugs, such as hormones, antibiotics, or the like, fixation of nitrogen, fermentation, utilization of specific feedstocks or the like. Genetic information for the biosynthesis of the enzyme is transferred to a microorganism wl1ich lacks the inorganic pyrophosphate acetate phosphotransferase conferring on this organism tl1e ability to grow on ;norganic pyrophosphate as shown in Example XIV, below; thus a capability for growth on pyrophosphate can be transferred to other microorganisms.

Example I

_sulfotomaculum nigrificans cells were grown on the_ _ _ following media: basal medium; basal medium plus inorganic pyrophosphate; basal medium minus sulfate plus inorganic pyrophosphate; basal medium minus acetate plus inorganic pyrophosphate; basal medium minus yeast extract plus inorganic pyrophosphate; basal medium minus acetate and yeast extract plus inorganic pyrophosphate; and lactate-sulfate medium.
The basal medium contained per liter: sodium acetate, 3.3 g;
Na2SO4, 0.4 gm; MgSO4 7H20, 0.2 g; MgCl26H2O, 1-8 gm; K2HPO4, 0.5 g; CaCl2 2H2O, 0.2 g; Difco Yeast Extract, 2.0 gm; FegO4, l0 mg; reducing agent (2.5 gm cystein HCl plus 2.5 gm Na2S
9H2O per 200 ml 112O) 20 ml. KOH was utilized to adjust the pH to 7.2. Where indicated, inorganic pyrophosphate (PPi) (filter sterilized) was added giving a final concentration of 0.05% in the medium per liter. The lactate-sulfate medium contained per liter: sod;um lactate (60%), 12.5 ml; NH4Cl, ,r ~z~

2.0 g; MgS04 7H20~ 0.2 g; K2HP04, 0.5 g; CaC12 2H20, 0-2g;
Difco Yeast Extract, 1.0 g; Na2S 9H20, 0.25 g.

Table 1, above, shows comparat;ve growth after forty-eight hours.

ExampleII

Desulfotomaculum ruminis cells were grown on the basal medium of Example I and with the addition of inorganic pyrophosphate (PPi) (filter sterilized) giving the following respective final concentrations in the medium per liter: 0.01%, 0.02%, 0.03%, 0.0~%, 0.05%, 0.06%, and 0.07%. Figure l shows comparative growth rates after ~orty-eight hours.

Example III

esulfotomaculum orientis cells were grown on basal medium plus inorganic pyrophosphate and lactate-sulfate medium both of Example I. Table 29 above, shows the comparative enzymatic activities.

Example IV

Mud samples from a salt water spartina marsh were used to inoculate the basal medium of Example I supplemented with sodium chloride giving a concentration of 2.5% in the medium per liter and the basal medium plus inorganic pyrophosphate of Example I supplemented with 2.5% sodium chloride. Photo-micrographs such as Figure 2 showed the diversity of cell-type after incubation anaerobically for twenty-four hours.

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Example V

Mud samples from a fresh water marsh were used to inoculate the basal medium of Example I and the basal medium plus inorganic pyrophosphate of Example I. After incubation anaerobically for twenty-four hours, the cultures exhibited the same extent of microbial diversity as observed with en-richment cultures from the salt water spartina marsh.

Example VI

Utilizing "state of the art media and growth conditions", inorganic pyrophosphate has been demonstrated to stimulate the physiological processes and growth of a number of diverse microorganisms. In Table 3, below, a list of microorganisms effected by inorganic pyrophosphate is presented.

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725~1 - --1 s--Table 3. The Effects of Pyrophosphate on the Growth of Various Microorganisms Growth Growth Electron Support- Stimulat- Acceptor ing ingFixed Carbon (Sink) _ . nigri-fic_ s + ....... Acetate; Sulfate Yeast Extract Dt. ruminis + ....... Acetate; Sulfate Yeast Extract Dt. orientis + ....... Acetate; Sulfate __ Yeast Extract Thiobacillus ? +None Nitrate denitrificans (Na S 0 ) 15 Methanobac-terium ? + Yeast Extract C02 Casitone 'rhermoauto-___ _ trophicum 20 Methanosarcina barkerii ? +Acetate; Acetate Yeast Extract (acetate Casitone methanol) 25 Clostridium thermocellum + ....... Yeast Extract .......
Rhodopseu_omona capsulata ? + Acetate .......
acetate 30 Thermoanaero-bacter _ hanolicus + ....... Yeast Extract None Desulfovibrio vulgaris - ....... ....... .......
Escherichia coli - ....... ....... ......

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Example VII

Mud from a fresh water marsh was employed to inoculate a cellulose contair.ing medium (Dilworth, G., Wiegel, J., Ljungdahl, L. G. and Peck, H. D., Jr., in Cellulose Microbienne, CMRS, Marseille, France. Cellulose (Avicel), 5.0 g/l;
NaHC03, 4.0 g/l; Yeast Extract, 0.6 g/l; Casitone, 2.5 g/l;
Cellobiose, 0.2 g/l; NaCl, 0.9 g/l; (NH4)2S04, 0.9 g/l; KH2P04, 0.45 g/l; MgS04, 0.09 g/l; CaCl2, 0.09 g/l; K2HP04, 0.45 g/l;
Cysteine, 0.5 g/l ) with and without 0.04% inorganic pyrophos-phate. The stimulation of the rate of methane formation is shown in Table 4, below.

Table 4. Stimulation of Methane Production from Cellulose by Inorganic Pyrophosphate with Enrichment Cultures Methane Formation (m moles) Days Minus Pyrophosphate Plus_Pyrop_osphate 7 0.03 0.06 9 0.04 0.15 ll 0.07 0.64 20 13 O.l5 2.04 0.24 2.67 17 0.38 3.63 Example VIII

Methanosarcina barklerii was inoculated into an acetate (0.2%) containing medium with and without 0.04% inorganic pyrophosphate. The stimulation in the rate of methane forma-tion by pyrophosphate is shown in Table 5, below.

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Table 5. The Stimulation of Methane. Formation from Acetate by Inorganic Pyrophosphate with Methanosarcina barkerii.
Methane Formation (m moles) 5 Days Minus Pyrophosphate Plus Pyrophosphate

3 0.18 0.26 0-77 1.23 7 1.40 3.92 9 1.63 6.38 10 ll 1.43 7.39 Example IX

A medium containing a fermentable compound such as glucose or starch is inoculated with a fermentative anaerobic bacterium; for example, Thermoanaerobacter ethanolicus with and without inorganic pyrophosphate and inoculated at the optimal growth temperature for the bacterium.

Example X

Samples of crushed low grade pyrite ores are inoculated with Thiobacillus uith and without inorganic pyrophosphate.
The release of metal ions is followed as a function of time at 30C. In the presence of inorganic pyrophosphate, there is an increased release of metal ions indicating increased growth of the Thiobacilli and an increase rate of leaching of the ore.

Example XI

Samples of pulverized high sulfur coal are inoculated with Thiobacillus with and without inorganic pyrophosphate.
The formation of sulfate ion is followed as a function of time at 30~C. In the presence of inorganic pyrophosphate, there is an increased production of sulfate indicating increased growthof theThiobacilli and an increased rate of desulfuri~ation of the coal samples.

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Example XII

Methan_ arcina barkerii is inoculated into a standard_ medium (Weimer, P. J. and Zeikus, J. G., Arch. Microbiol.
119:46-57, 1978. KH2P04, 1.5 g/985 ml glass distilled water;
K2HP04.3H20, 2.9 g/985 ml glass distilled water; NH4Cl, l.0 g/985 ml glass distilled water; NaCl, 0.9 g/985 ml glass distilled water; MgC12.6H20, 0.2 g/985 ml glass distilled water; CaCl2.2H20, 0.05 g/985 glass ml distilled water;
NaSeO3, 0.017 mg/985 ml glass distilled water; Mineral 10 Solution, lO ml/985 ml glass distilled water; Vitamin Solution, 5 ml/985 ml glass distilled water; Reazurin (0.2%), l.0 ml/985 ml glass distilled water ) containing inorganic pyrophosphate.
Increased yields of vitamin B12 are obtained such that the organism can be utilized for the commercial production of the 15 vitamin.

Example XIII

Clostridium thermocellum is inoculated into a standard_ medium (Dilworth, G., Wiegel, J., Ljungdahl, L. G. and Peck, H. D., Jr.~ in Cellulose Microbienne, CMRS, Marseille, France.
20 Cellulose (A~icel), 5.0 g/l; NaHC03, 4.0 g/l; Yeast Extract, 0.6 g/l; Casitone, 2.5 g/l; Cellobiose, 0.2 g/l; NaCl, 0.9 g/l; ~NH4)2S04, 0.9 gJl; KH2P04, 0.45 g/l; MgS04, 0.09 g/l;
CaCl2, 0.09 gel; K2HP04, 0.45 g/l; Cysteine, 0.5 g/l ) supple-mented with inorganic pyrophosphate. Increased growth of 25 _ ostridium thermocellum is obtained.

Example XI~

Escherichia coli is unable to grow on inorganic pyrophos-phate and lacks the enzyme, pyrophosphate acetate phosphotrans-ferase. DNA from Desulfotomaculum containing the information 30 for the biosynthesis of the phosphotransferase is transferred to E. coli using techniques known in the art for the transfer of deoxyribonucleic acid (DNA) from one microorganism to another, U.S. Patent No. 4,237,224 (Cohen and Boyer, Process :IL2~7~51~

for Producing Biologically Functional Molecular Chimeras is a process which is known in the art. Method and compositions are provided for replication and expression of exogenous genes in microorganisms. Plasmids or virus deoxyribonucleic acid are cleaved to provide a biologically functional replicon with a desired phenotypical property. The replicon is inserted into a microorganism cell by transformation. Isolation of the transformants provides cells for replication and expression of the deoxyribonucleic acid molecules present in the modified plasmid. The method provides a convenient and efficient way to introduce genetic capability into microorganisms for the production of nucleic acids and proteins, such as medically or commercially useful enzymes, which may have direct usefulness, or may find expression ;n the production of drugs, such as hormones, antibiotics, or the like, fixation of nitrogen, fermentation utilization of specific feedstocks, or the like ) allowing the microorganism Jo utilize inorganic pyrophosphate as a source of energy for growth.

The foregoing illustrates specific embodiments within the scope of this invention and is not to be construed as limiting said scope. While the invention has been described herein with regard to a certain specific embodiment, it is not so limited. It is to be understood that variations and modifi-cations thereof may be made by those skilled in the art without departing from the scope of the invention.

Indust_ al Applicabilit This invention is a process for using inorganic pyrophos-phate as an energy source to stimulate growth rates of micro-organisms used in commercial and industrial applications such as leaching of low grade pyrite ores, desulfurization of coal, conversion of biomass to ethanol, and conversion of biomass to methane.

Claims (21)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for cultivating a microorganism, which comprises:
cultivating a microorganism in a growth medium containing an inorganic pyrophosphate wherein said micro-organism is capable of utilizing said inorganic pyrophosphate as an energy source for internal generation of adenosine triphosphate when said inorganic pyrophosphate is externally supplied to said microorganism.

2. A process according to Claim 1 wherein the micro-organism is selected from the group consisting of Desulfoto-maculum species, Methanobacterium species, Methanosarcina species, Thermoanaerobacter species, Thiobacillus species, and Clostridium species.

3. A process according to Claim 2 wherein the microorganism produces pyrophosphate acetate phosphotransferase, and acetate kinase at levels sufficient to allow utilization of the inorganic pyrophosphate as an energy source.

4. A process according to Claim 1 wherein the growth medium comprises acetate and yeast extract.

5. A process according to Claim 1 wherein the growth medium contains an electron sink for adjusting the oxidation level of a fixed carbon source.

6. A process according to Claim 5 wherein the electron sink is sulfate.

7. A process according to Claim 1 wherein the concentration of inorganic pyrophosphate in said growth medium is between 0.01% (w/v) and 0.06% (w/v).

8. A process for the stimulation of the growth rate of a microorganism, which comprises:
adding an inorganic pyrophosphate to a composition which comprises a microorganism in a growth medium which does not contain said inorganic pyrophosphate wherein said microorganism is capable of utilizing said inorganic pyro-phosphate as an energy source for internal generation of adenosine triphosphate when said inorganic pyrophosphate is externally supplied to said microorganism.

9. A process according to Claim 8, wherein the microorganism is selected from the group consisting of Desulfotomaculum species, Methanobacterium species, Methanosarcina species, Thermoanaerobacter species, Thiobacillus species, and Clostridium species.

10. A process according to Claim 9 wherein the microorganism produces pyrophospate acetate phosphotranserase and acetate kinase at levels sufficient to allow utilization of the inorganic pyrophospate as an energy source.

11. A process according to Claim 8 wherein the microorganism converts cellulose to methane and carbon dioxide.

12. A process according to Claim 8 wherein the microorganism converts biomass to methane and carbon dioxide,

13. A process according to Claim 8 wherein the microorganism converts cellulose to ethanol and other products.

14. A process according to Claim 8 wherein the microorganism converts biomass to ethanol and other products.

15. A process according to Claim 8 wherein the microorganism leaches low grade pyrite ores.

16. A process according to Claim 8 wherein the microorganism desulfurizes coal.

17. A process according to Claim 8 wherein the microorganism is used to produce an enzyme.

18. A process according to Claim 8 wherein the microorganism is used to produce a compound selected from the group consisting of fine biochemicals and pharmaceuticals.

19. A process according to Claim 18 wherein the microorganism is Methanosarcina barkerii and wherein the fine biochemical is vitamin B12.

20. A process according to Claim 8 wherein genetic information for a pyrophosphate acetate phosphotransferase enzyme was transferred to the microorganism using techniques known in the art for the transfer of deoxyribonucleic acid from one microorganism to another.

21. A process according to Claim 20 wherein the micro-organism is Escherichia coli.