WO1983002952A1

WO1983002952A1 - Stimulation of bacterial growth by inorganic pyrophosphate

Info

Publication number: WO1983002952A1
Application number: PCT/US1983/000227
Authority: WO
Inventors: Jr. Harry D. Peck; Nancy K. Hart; Chi-Li Liu; Jean Legall
Original assignee: University Of Georgia Research Foundation, Inc.
Priority date: 1982-02-26
Filing date: 1983-02-22
Publication date: 1983-09-01
Also published as: JPS6027511B2; EP0101721A1; JPS59500253A; EP0101721A4

Abstract

A process for the growth of microorganisms wherein inorganic pyrophosphate is used as an energy source to generate adenosine triphosphate. Microorganisms with the enzymes acetate phosphotransferase and acetate kinase 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

STIMULATION OF BACTERIAL GROWTH BY INORGANIC PYROPHOSPHATE

DESCRIPTION

This application claims a priority filing date based on an application, entitled "Stimulation of Bacterial Growth by Inorganic Pyrophosphate," Serial No. 352,742, filed February 26, 1982 (02.26.82), in the United States Patent and Trademark Office as a national application.

The Government has rights in this invention pursuant to Con tract No. DEAS 09-79 ER 10499 awarded by the U.S. Department of Energy.

Technical Field

This invention relates to the growth of microorganisms using inorganic pyrophosphate as an energy source. This invention over comes low or slow growth problems in many species of microorganisms, particularly species used in important commercial and industrial processes.

Background Art

Inorganic pyrophsophate (PP_i) 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., 77:1927, 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. 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 (PP.), produced from adenosine triphosphate (ATP) by the enzyme adenosine triphosphate sulfurylase (Eq. 1) in the first enzymatic step of

Eq. 1 ATP + SO₄ ^-2 →ATP + PP_i

respiratory sulfate reduction, is hydrolyzed to orthophosphate (P_i) by inorganic pyrophosphatase (Eq. 2).

Eq. 2 PP_i + H₂O →2P_i

Thus, the chemical energy in the anhydride bond of inorganic pyrophosphate (PP_i) is not conserved and, in order to obtain a net yield of adenosine triphosphate (ATP) during growth on a lactatesulfate medium, Desulfovibrio species carry out electron transfer 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. Gutherie, Biochem. Biophys. Res. Commun., 66: 1389, 1975).

Eq. 3 Acetate + PPi →Acetylphosphate + P_i

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 These two enzymatic reactions allow Desulfotomaculum species to generate one high energy phosphate by substrate level phosphoryla tion per sulfate reduced to sulfide during growth with lactate on the lactate-sulfate medium. It is not necessary for Desulfotomaculum species to carry out electron transfer coupled phosphoryla tion 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, utilization of inorganic pyrophos phate as an energy source represents overall the simplest adenosine triphosphate generating system in the biological world requiring only one specific enzyme, phyrophosphate acetate phosphotransferase, plus the ubiquitous, acetate kinase.

From the standpoint of microbial ecology, the widespread occurrence of inorganic pyrophosphate utilization suggests that inorganic pyrophosphate functions as a new type of energy transfer system. The demonstration of inorganic pyrophosphate as an important part of the phosphorus cycle will represent a major contribution to the understanding of microorganisms and their relation- ships 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 microbiology, and inorganic pyrophosphate is a common and inexpensive chemical.

The present invention is a process for the growth of microorganisms wherein inorganic pyrophosphate is used as an energy source.

It is an object of the present invention to provide a process for using inorganic pyrophosphate, in the presence of fixed carbon, as a source of adenosine triphosphate for growth. These and other objects, aspects, and advantages of this invention will become apparent from a consideration of the accompanying specification and claims. It is a further object to provide a process for using inorganic pyrophosphate as an energy source to stimulate growth rates of microorganisms used in commercial and industrial applications such as leaching of low grade pyrite ores, desulfurization of coal, conversion of biomass to ethanol, and conversaion of biomass to methane.

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 photomicrographs of microorganisms growing in inorganic pyrophosphate (PP_i) enrichments of marine mud using the basal medium supplemented with 2.5% sodium chloride as described in Example IV, below..

Modes for Carrying Out the Invention

Desulfotomaculum nigrificans, Desulfotomaculum ruminis, and Desulfotomaculum 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 I, below, compares growth on various media. The basal medium, containing acetate, yeast extract, sulfate, and salts does not 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 orthophosphate, equivalent to the added inorganic pyrophosphate, does not support the growth of Desulfotomaculum nigrificans, Desulfotomaculum ruminis, and Desulfotomaculum orientis. For optimal growth of Desulfoto- maculum nigrificans on inorganic pyrophosphate, acetate, yeast extract, and sulfate were required; acetate and yeast extract were the fixed carbon source, 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 concentration of sulfate was only 1/10 of that used in the usual lactate-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 of the permeation of acetate into Desulfotomaculum nigrificans. 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 ratio of 1:1. Similar growth responses were found for Desulfotomaculum ruminis and Desulfotomaculum orientis; therefore, inorganic pyrophosphate served as an energy source for growth of these anaerobic sulfate reducing microorganism.

Table 1. Requirements for the Growth of Desulfotomaculum

Utilizing Inorganic Pyrophosphate (PP_i) as a Source of Energy

Additions and/or Deletions Growth* (O.D.)

Basal Medium 0.019

Basal Medium plus PP_i 0.628

Basal Medium minus Sulfate plus PP_i 0.019

Basal Medium minus Acetate plus PP_i 0.095 Basal Medium minus Yeast Extract plus PP_i 0.042

Basal Medium minus Acetate and Yeast Extract plus PP_i 0.036

Lactate-Sulfate Medium 0.505 *Measured at 580 nm; average of duplicate flasks after forty eight hours incubation at 55ºC under argon.

The date for Example II, below, illustrated in Figure 1 shows the growth response of Desulfotomaculum ruminis to increasing amounts of inorganic pyrophosphate. The growth response was proportional to inorganic pyrophosphate concentrations 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 (pH 8.5) as a result of inorganic pyrophosphate hydrolysis. Similar results were obtained with Desulfotomaculum nigrificans and Desulfotomaculum orientis.

The enzymatic complement of cells of Desulfotomaculum orientis 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 lactatesulfate grown cells of Desulfotomaculum 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. 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 dehy drogenase 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 Desulfotomaculum nigrificans and Desulfotomaculum rumi- nis 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. Bacter iol. 147: 161-169, 1981. Enzyme assays. Benzyl viologen- or meth yl viologen-coupled nitrite, sulfite, fumarate, and thiosulfate reductases and hydrogenase were all assayed manometrically. Reaction mixtures consisted of 100 mM (pH 7.4) phosphate, 5.0 mM benzyl or methyl viologen, and 20.0 mM sulfite, 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.vul garis (van der Westen, H., S. G. Mayhew, and C. Veeger, FEBS Lett.

86: 122-126, 1978) was added in all manometric viologen-linked assays except the hydrogenase assay. Pyruvate-BV²⁺ and formateBV²⁺ reductases were determined spectrophotometrically at 545 nm in a Beckman model 25 spectrophotometer. Reaction mixtures contained 5.0 mM BV²⁺, 10.0 mM dithiothreitol, 100 mM potassium phosphate buffer (pH 7.4), and 20 mM sodium formate for the formate BV²⁺ reaction and 5.0 mM BV²⁺, 2.0 mM reduced coenzyme A, 100 mM potassium phosphate (pH 7.4), and 20.0 mM sodium pyruvate for the pyruvate-BV²⁺ 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-1.0 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-10mM ferricyanide-100 mM potassium phosphate, pH 7.4, for the succinate-ferricyanide 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 O₂oxidized and dithionate-reduced membranes were measured at liquid nitrogen temperatures in an Aminco DW-2 spectrophotometer with a 1-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 (M_r = 13,000) 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 Enzymol. 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 (Gornall, A. G. , G. J. Bardawill, and M. M. David, J. Biol. Chem. 177:751-766, 1949). Liu, Chi-Li and Peck, H. D. Jr., J. Bacteriol. 145:466-673, 1981.

Assays. Adenosine triphosphate sulfurylase was determined with MoO₄ ^2- as described by Wilson and Bandurski (Wilson, L. G. and R.

S. Bandurski, J. Biol. Chem. 233:975-981, 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. Brauer, 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.)

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 microorganisms. 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 37ºC the anaerobic medium containing inorganic pyrophosphate showed extensive microbial growth, and photomicrographs of the microorganisms in such enrichments were made. The photomicrographs of these enrichment cultures contained a surprising high and unexpected number of morphological types of mircoorganisms. 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 an energy 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, Methanobacterium species, and Methanosarcina species are directly involved in the microbial community responsible for the conversion of cellulose, including biomass and organic wastes, to methane and carbon dioxide. Desulfotomaculum species convert fermentation products such as lactate, fatty acids and alcohols to acetate, carbon dioxide and hydrogen; Methanobacterium species convert carbon dioxide and hydrogen to methane; and Methanosarcina species convert acetate to methane and carbon dioxide. The conversion of fermentation products and pro- duction of methane from acetate or hydrogen plus C0„ 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 ethanolicus, a thermophilic fermentative 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 ethanolicus for the continuous production of ethanol. The addition of inorganic pyrophosphate removes, reduces, or eliminates the formation of acetate and lactate and thereby facilitating the continuous production of ethanol by Thermoanaerobacter ethanolicus as shown in Example IX, below.

Thiobacillus 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 microorganisms as shown in Example X, below. A reduced residence time increases the capacity of existing facilities used in this process. A second aspect of microbial leaching by Thiobacillus species additions of inorganic pyrophosphate accelerate the desulfurization process by increasing the initial growth rates of these microorganisms thereby providing the increased bacterial mass required for the process. A reduced residence time for the coal slurry makes the process economically viable.

Microbial processes are utilized for the industrial production of a large number of enzymes, fine biochemicals, and pharmaceuticals. Addition of inorganic pyrophosphate to production media increases the yield of these products and microorganism. For examples, the yield of vitamin B₁₂ by Methanosarcina barkerii is increased by the addition of inorganic pyrophosphate as shown in Example XII, below; and the amount of cellulase produced by Clostridium thermocellum is increased by the addition of inorganic pyrophosphate as shown in Example XIII, below. The inorganic pyrophosphate acetate phosphotransferase represents the simplest biological system for producing ATP from a substrate. Using "state of the art" techniques for the transfer of deoxyribonucleic acid (DNA) from one microorganism to another, U. S. Patent No. 4,237,224 (Cohen and Boyer, Process 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 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 which lacks the inorganic pyrophosphate acetate phosphotransferase conferring on this organism the ability to grow on inorganic pyrophosphate as shown in Example XIV, below; thus a capability for growth on pyrophosphate can be transferred to other microorganisms.

Example I

Desulfotomaculum 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 7H₂O, 0.2 g; MgCl₂6H₂O, 1.8 gm; K₂HPO₄, 0.5 g; CaCl₂ 2H₂0, 0.2 g; Difco Yeast Extract, 2.0 gm; FeSO₄, 10 mg; reducing agent (2.5 gm cystein HCI plus 2.5 gm Na₂S 9H₂O per 200 ml H₂O) 20 ml. KOH was utilized to adjust the pH to 7.2. Where indicated, inorganic pyrophosphate (PP.) (filter sterilized) was added giving a final concentration of 0.05% in the medium per liter. The lactate-sulfate medium contained per liter: sodium lactate (60%), 12.5 ml; NH₄C1, 2.0 g; MgSO₄ 7H₂O, 0.2 g; K₂HPO₄, 0.5 g; CaCl₂ 2H₂O, 0.2 g; Difco Yeast Extract, 1.0 g; Na₂S 9H₂O, 0.25 g.

Table 1, above, shows comparative growth after forty-eight hours.

Example II

Desulfotomaculum ruminis cells were grown on the basal medium of Example I and with the addition of inorganic pyrophosphate (PP.) (filter sterilized) giving the following respective final concentrations in the medium per liter: 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, and 0.07%. Figure 1 shows comparative growth rates after forty-eight hours.

Example III

Desulfotomaculum orientis cells were grown on basal medium plus inorganic pyrophosphate and lactate-sulfate medium both of Example I. Table 2, 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. Photomicrographs such as Figure 2 showed the diversity of cell-type after incubation anaerobically for twenty-four hours.

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 enrichment 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.

Example VII

Mud from a fresh water marsh was employed to inoculate a cellulose containing medium (Dilworth, G., Wiegel, J., Ljungdahl, L. G. and Peck, H. D., Jr., in Cellulose Microbienne, CMRS, Marseille, France. Cellulose (Avicel), 5.0 g/1; NaHCO₃, 4.0 g/1; Yeast Extract, 0.6 g/1; Casitone, 2.5 g/l; Cellobiose, 0.2 g/l; NaCl, 0.9 g/1; (NH₄)₂SO₄, 0.9 g/l; KH₂PO₄, 0.45 g/l; MgSO₄, 0.09 g/l; CaCl₂, 0.09 g/l; K₂HPO₄, 0.45 g/l; Cysteine, 0.5 g/l.) with and without 0.04% inorganic pyrophosphate. 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

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

Table 5. The Stimulation of Methane. Formation from Acetate by

Inorganic Pyrophosphate with Methanosarcina barkerii.

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. In the presence of inorganic pyrophosphate the fermentation products are altered such that a higher concentration of the desired product is obtained.

Example X

Samples of crushed low grade pyrite ores are inoculated with Thiobacillus with and without inorganic pyrophosphate. The release of metal ions is followed as a function of time at 30ºC. 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 30ºC. In the presence of inorganic pyrophosphate, there is an increased production of sulfate indicating increased growth of the Thiobacilli and an increased rate of desulfurization of the coal samples. Example XII

Methanosarcina barkerii is inoculated into a standard medium (Weiner, P. J. and Zeikus, J. G. , Arch. Microbiol. 119:46-57, 1978. KH₂PO₄, 1.5 g/985 ml glass distilled water; K₂HPO₄.3H₂O, 2.9 g/985 ml glass distilled water; NH.Cl, 1.0 g/985 ml glass distilled water; NaCl, 0.9 g/985 ml glass distilled water; MgCl₂. 6H 0, 0.2 g/985 ml glass distilled water; CaCl₂.2H₂O, 0.05 g/985 glass ml distilled water; NaSeO₃, 0.017 mg/985 ml glass distilled water; Mineral Solution, 10 ml/985 ml glass distilled water; Vitamin Solution, 5 ml/985 ml glass distilled water; Reazurin (0.2%), 1.0 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 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. Cellulose (Avicel), 5.0 g/1; NaHCO , 4.0 g/1; Yeast Extract, 0.6 g/1; Casitone, 2.5 g/1; Cellobiose, 0.2 g/1; NaCl, 0.9 g/1; (NH₄)₂SO₄, 0.9 g/1; KH₂PO₄, 0.45 g/1; MgSO₄, 0.09 g/1; CaCl₂, 0.09 g/1; K₂HPO₄, 0.45 g/1; Cysteine, 0.5 g/1.) supplemented with inorganic pyrophosphate. Increased yields of cellulose are obtained such that C. thermocellum can be utilized for the commercial production of cellulase.

Example XIV

Escherichia coli is unable to grow on inorganic pyrophosphate and lacks the enzyme, pyrophosphate acetate phosphotransferase. DNA from Desulfotomaculum containing the information for the biosnythesis 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 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 vi rus 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 in 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 to 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 modifications thereof may be made by those skilled in the art without departing from the scope of the invention.

Industrial Applicability This invention is a process for using inorganic pyrophos phate as an energy source to stimulate growth rates of microorganisms 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

CLAIMS What is claimed is:

1. A process for the growth of microorganisms wherein inorganic pyrophosphate is used as an energy source for generating adenosine triphosphate on a medium containing a fixed carbon source and wherein the microorganisms have pyrophosphate acetate phosphotransferase enzyme and an acetate kinase enzyme.

2. A process according to claim 1 wherein the microorganisms are from mud samples of anaerobic ecosystems selected from the group consisting of a salt water spartina marsh, a fresh water marsh, sewerage sludge, and a rumen.

3. A process according to claim 1 wherein the microorganisms are aerobic.

4. A process according to claim 1 wherein the microorganisms are anaerobic.

5. A process according to claim 4 wherein the microorganisms are non-sulfate reducing.

6. A process according to claim 4 wherein the microorganisms are sulfate reducing.

7. A process according to claim 4 wherein the microorganisms are selected from the group consisting of Desulfotomaculum species, Methanobacterium species, Methanosarcina species, Thermoanaerobacter species, Thiobacillus species, and Clostridium species.

8. A process according to claim 5 wherein the microorganisms are Thermoanaerobacter ethanolicus.

9. A process according to claim 6 wherein the microorganisms are selected from the group consisting of Desulfotomaculum nigrificans, Desulfotomaculum orientis, and Desulfotomaculum ruminis.

10. A process according to claim 9 wherein the fixed carbon source comprises acetate and yeast extract.

11. A process according to claim 10 wherein the medium contains an electron sink for adjusting the oxidation level of the fixed carbon source.

12. A process according to claim 11 wherein the electron sink is sulfate.

13. A process according to claim 10 wherein the concentration of inorganic pyrophosphate is between 0.1% (w/v) and 0.06% (w/v).

14. A process according to claim 10 wherein the concentration of inorganic pyrophosphate is 0.05% (w/v).

15. A process according to claim 6 wherein the microorganisms are Methanosarcina barkerii.

16. A process for the stimulation of growth rates of microorganisms wherein inorganic pyrophosphate is used as an energy source for generating adenosine triphosphate on a medium containing a fixed carbon source and wherein the microorganisms have a pyrophosphate acetate phosphotransferase enzyme and an acetate kinase enzyme.

17. A process according to claim 16 wherein the microorganisms convert cellulose to methane and carbon dioxide.

18. A process according to claim 16 wherein the microorganisms convert biomass to methane and carbon dioxide.

19. A process according to claim 16 wherein the microorganisms convert cellulose to ethanol and other products.

20. A process according to claim 16 wherein the microorganisms convert biomass to ethanol and other products.

21. A process according to claim 16 wherein the microorganisms leach low grade pyrite ores.

22. A process according to claim 16 wherein the microorganisms desulfurize coal.

23. A process according to claim 16 wherein the microorganisms are used to produce an enzyme.

24. A process according to claim 23 wherein the microorganisms are Clostridium thermocellum and wherein the enzyme is cellulase.

25. A process according to claim 16 wherein the microorganisms are used to produce compounds selected from the group consisting of a fine biochemical and a pharmaceutical.

26. A process according to claim 25 wherein the microorganisms are Methanosarcina barkerii and wherein the fine biochemical is vitamin B₁₂.

27. A process according to claim 16 wherein genetic information for the pyrophosphate acetate phosphotransferase enzyme was transferred to the microorganisms using techniques known in the art for the transfer of deoxyribonucleic acid (DNA) from one microorganism to another.

28. A process according to claim 27 wherein the microorganisms are Escherichia coli.