DESCRIPTION

This application claims a priority filing date based on an application, entitled "Stimulation of Bacterial Growth by Inorgan¬ ic 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 microorgan¬ isms, particularly species used in important commercial and indus¬ trial processes.

Background Art

Inorganic pyrophsophate (PP.) 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., _^ 43:85, 1977; K. S. Lam, C. B. Kasper, Proc. Natl. Acad. Sci., 7_7:1927, 1980; N. W. Carnal, C. C. Black, Biochem. Biophys. Res. Commun. , 36_: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 pyro- phosphate 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 Desulfo omaculu , are fundamentally different (C. L. Liu, H. D. Peck, Jr., J. Bacteriol. , 145:966, 1981). In the case of Desul¬ fovibrio,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 + S0 ₄ ~ ATP + PP _±

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

Eq. 2 PP. + H _o 0 ^2P.

^ l 2 x

Thus, the chemical energy in the anhydride bond of inorganic pyro¬ phosphate (PP.) is not conserved and, in order to obtain a net yield of adenosine triphosphate (ATP) during growth on a lactate- sulfate medium, Desulfovibrio species carry out electron transfer coupled phosphorylation in this growth mode. In contrast, Desul- fotomaculum species are able to conserve the bond energy of the pyrophosphate produced by adenosine triphosphate (ATP) sulfuryl¬ ase (Eq. 1) by means of the enzyme, inorganic pyrophosphate ace¬ tate phosphotransferase (Eq. 3) (R. E. Reeves, J. B. Gutherie, Biochem. Biophys. Res. Commun., 66:1389, 1975).

Eq. 3 Acetate + PP.- ^ cetyl phosphate + P.

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 v

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 5 the lactate-sulfate medium. It is not necessary for Desulfotoma¬ culum 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 10 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 sulf te-reducing species belonging to the genus Desulfotomaculum, utilization of inorganic pyrophos- 15 phate as an energy source represents overall the simplest adeno¬ sine triphosphate generating system in the biological world requi¬ ring only one specific enzyme, phyrophosphate acetate phosphotran¬ sferase, plus the ubiquitous, acetate kinase.

From the standpoint of microbial ecology, the widespread 20. 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 impor¬ tant part of the phosphorus cycle will represent a major contribu¬ tion to the understanding of microorganisms and their relation- 5 ships in different ecosystems such as the salt water marsh and sediments, fresh water marsh and sediments, anaerobic sludge di- gestors, 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 0 inexpensive chemical.

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

It is an object of the present invention to provide a process 5 for using inorganic pyrophosphate, in the presence of fixed car¬ bon, 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 accom¬ panying specification and claims. It is a further object to provide a process for using inor¬ ganic 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 bio- mass to methane.

Brief Description of the Drawings

Figure 1 shows the effect of inorganic pyrophosphate concen¬ tration on the growth of Desulfotomaculum ruminis. Growth condi¬ tions were the same as in Example II, below, using the basal medi- um and with varying concentrations of inorganic pyrophosphate. Figure 2 shows two photomicrographs of microorganisms growing in inorganic pyrophosphate (PP.) 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 inor¬ ganic pyrophosphate, acetate, yeast extract, sulfate, and salts. In the sulfate reducing Desulfotomaculum species of microorgani- s s, the use of inorganic pyrophosphate as an energy source in a process for growth requires only one specific enzyme, pyrophos¬ phate acetate phosphotransferase. The ubiquitous enzyme, acetate kinase, is also required. Adenosine triphosphate is generated by this system. Also, crude enrichment cultures from a marine spar- tina marsh and fresh water marshes were similarly grown using in¬ organic 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 medi- um, 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. 5 On the basal medium, inorganic pyrophosphate does not stimulate the growth of Desulfovibrio vulgaris; and orthophosphate, equiva¬ lent to the added inorganic pyrophosphate, does not support the growth of Desulfotomaculum nigrificans, Desulfotomaculum ruminis, and Desulfotomaculum orientis. For optimal growth of Desulfoto-

10 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

15 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 physi¬ ological basis for this high acetate requirement may have involved the bioenergetics of the permeation of acetate into Desulfotoma-

20. culu nigrificans. The stimulatory effect of inorganic pyrophos¬ phate 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 5 were found for Desulfotomaculum ruminis and Desulfotomaculum ori¬ entis; therefore, inorganic pyrophosphate served as an energy source for growth of these anaerobic sulfate reducing microorgan¬ ism.

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■ _v -r/ - WIFO

Table 1. Requirements for the Growth of Desulfotomaculum

Utilizing Inorganic Pyrophosphate (PP.) as a Source of Energy *

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

Basal Medium 0.019

Basal Medium plus PP. 0.628

Basal Medium minus Sulfate plus PP. 0.019

Basal Medium minus Acetate plus PP. 0.095 Basal Medium minus Yeast Extract plus PP. 0.042

Basal Medium minus Acetate and Yeast Extract plus PP. 0.036 ^r x

Lactate-Sulfate Medium 0.505

-__ Measured at 580 nm; average of duplicate flasks after forty- o eight hours incubation at 55 C 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 pro¬ portional to inorganic pyrophosphate concentrations up to 0.04% and growth was accompanied by the hydrolysis of inorganic pyro¬ phosphate. In the absence of growth, there was little hydrolysis of added inorganic pyrophosphate. Above 0.05%, inorganic pyro¬ phosphate growth was inhibited which may have been due to alkali- zation of the medium (pH 8.5) as a result of inorganic pyrophos- phate hydrolysis. Similar results were obtained with Desulfoto¬ maculum nigrificans and Desulfotomaculum orientis.

The enzymatic complement of cells of Desulfotomaculum orien¬ tis 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 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- 5 drogenase were present at similar specific activities. The reason for the significantly higher hydrogenase in inorganic pyrophos¬ phate grown cells may have been due to difficulties with the assay procedure. The unique occurrence of these enzymes was also con¬ irmed for Desulfotomaculum nigrificans and Desulfotomaculum rumi- 10 nis which indicated that the cells grown on inorganic pyrophos¬ phate exhibit no basic changes in their metabolic pattern.

Enzymatic activities were determined by the following stan¬ dard assay procedures (Odom, J. M. and Peck, H. D. Jr. , J. Bacter- iol. 147: 161-169, 1981. Enzyme assays. Benzyl viologen- or eth- 15 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 thio¬ sulfate or 5.0 mM nitrite in a total volume of 1.0 ml. Partially 20. 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

2+ assays except the hydrogenase assay. Pyruvate-BV and formate-

2+ BV reductases were determined spectrophotometrically at 545 nm 5 in a Beckman model 25 spectrophotometer. Reaction mixtures con-

2+ tained 5.0 mM BV , 10.0 M dithiothreitol, 100 mM potassium phosphate buffer (pH 7.4), and 20 mM sodium formate for the for-

2+ 2+ mate BV 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

2+ 0 the pyruvate-BV reaction. Both assays were performed under ar¬ gon in Thunberg covettes. Succinate-ferricyanide reductase and APS reductase assays were performed with a spectrophotometer, un¬ der 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, 5 for the APS reductase reaction (Bramlett, R. and H. D. Peck, Jr., J. Biol. Chem. 250:2979-2986, 1975) and 20.0 mM potassium succin- ate-10 mM 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 0„- 5 oxidized and dithionate-reduced membranes were measured at liquid nitrogen temperatures in an A inco DW-2 spectrophotometer with a 1-mm light path. Changes in the apparent extinction coefficients at liquid nitrogen temperatures were taken into account by measur¬ ing the difference spectra of known amounts of cytochrome c„ (M =

10 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 dinucleot de and flavin mononucleotide were determined by fluorescence at acid and neutral pH (Siegal, L. M. , Methods Enzy-

15 mol. 53D:419-429, 1979). Menaquinone was determined by the method of Kroger (Kroger, A., Methods Enzy ol. 53D:579-591, 1979), and non-heme iron was assayed by measuring color formation with o-phe- nanthroline (Beinert, H. , Anal. Biochem. 20:325-334, 1967). Pro¬ tein was determined by the biuret method (Gornall, A. G. , G. J.

20. 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

2- Mo0, as described by Wilson and Bandurski (Wilson, L. G. and R.

S. Bandurski, J. Biol. Chem. 233:975-981, 1958), and inorganic 25 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, 0 1975), except that acetyl phosphate (Lipmann, F. and L. C. Tuttle, J. Biol. Chem. 159:21-28, 1945) produced from acetate plus pyro¬ phosphate was determined. Sulfide was measured by the method of Siegel (Siegel, L. M. , Anal. Biochem. _L1:126-32, 1965), and pro¬ tein was measured by the biuret method (Levin, R. and R. W. Brauer, 35 J. Lab. Clin. Med. _3.:474-479, 1951.) Lactate and acetate were measured with a Varian Aerograph 2700 gas chromatograph equipped with a hydrogen flame ionization detector.)

Table 2. Enzymatic Activities in Extracts of Desulfotomacu¬ lum orientis Grown on Basal Medium plus Inorganic Pyrophosphate (PP.) 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 Reductase 119.6 153.8

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

Adenosine Triphosphate

Sulfurylase 140 151

Pyrophosphate

Acetate Phosphotransferase 820 1315

Inorganic Phyrophosphatase 149 98

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 microorgan- isms of other genera on basal medium plus inorganic pyrophosphate was tested using enrichment cultures of microorganisms. Specifi¬ cally, microorganisms from mud samples obtained from a salt water spartina marsh were utilized to inoculate the basal medium supple¬ mented with sodium chloride with and without inorganic pyrophos- phate under anaerobic conditions as described in Example IV, be¬ ta low. Within twenty-four hours at 37 C the anaerobic medium con¬ taining inorganic pyrophosphate showed extensive microbial growth, and photomicrographs of the microorganisms in such enrichments were made. The photomicrographs of these enrichment cultures con- tained a surprising high and unexpected number of morphological

OMFI '

types of ircoorganisms. Similar results were obtained with inor¬ ganic pyrophosphate enrichment grown cultures from fresh water en¬ vironments utilizing the same medium minus sodium chloride as 5 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 enrich¬ ment cultures were non-sulfate reducing microorganisms and did not

10 require acetate or sulfate for growth.

Desulfotomaculum species, Methanobacter um species, and Meth¬ anosarcina species are directly involved in the microbial communi¬ ty responsible for the conversion of cellulose, including biomass and organic wastes, to methane and carbon dioxide. Desulfotomacu-

15 lum species convert fermentation products such as lactate, fatty acids and alcohols to acetate, carbon dioxide and hydrogen; Metha- nobacterium species convert carbon dioxide and hydrogen to meth¬ ane; and Methanosarcina species convert acetate to methane and carbon dioxide. The conversion of fermentation products and pro-

20. duction of methane from acetate or hydrogen plus C0„ both, as des¬ cribed above, are limiting steps in this important biological pro¬ cess and the growth or physiological processes characteristic of these bacteria are stimulated by inorganic pyrophosphate as des¬ cribed in Example IV, below. The stimulation of methane formation

25 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

30 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 metabo¬ lize inorganic pyrophosphate suggests a process wherein inorganic

35 pyrophosphate is used to modify or alter the pattern of fermenta¬ tion 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 pyro¬ phosphate 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 in¬ creasing the initial growth rates of these microorganisms as shown in Example X, below. A reduced residence time increases the capa¬ city of existing facilities used in this process. A second aspect of microbial leaching by Thiobacillus species additions of inorganic pyrophosphate accelerate the desulfuriza¬ tion process by increasing the initial growth rates of these mi¬ croorganisms thereby providing the increased bacterial mass re¬ quired for the process. A reduced residence time for the coal slurry makes the process economically viable.

Microbial processes are utilized for the industrial produc¬ tion of a large number of enzymes, fine biochemicals, and pharma¬ ceuticals. Addition of inorganic pyrophosphate to production me¬ dia increases the yield of these products and microorganism. For examples, the yield of vitamin B. „ by Methanosarcina barkerii is increased by the addition of inorganic pyrophosphate as shown in Example XII, below; and the amount of cellulase produced by Clos- tridium thermocellum is increased by the addition of inorganic pyrophosphate as shown in Example XIII, below. The inorganic pyrophosphate acetate phosphotransferase repre¬ sents the simplest biological system for producing ATP from a sub¬ strate. 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 re¬ plication 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 mole¬ cules 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 pyro¬ phosphate 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 follow¬ ing media: basal medium; basal medium plus inorganic pyrophos¬ phate, 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 pyrophos¬ phate; and lactate-sulfate medium. The basal medium contained per liter: sodium acetate, 3.3 g; Na„S0,, 0.4 gm; MgSO, 7H„0, 0.2 g; MgCl ₂ 6H ₂ 0, 1.8 gm; K _£ HP0 ₄ , 0.5 g; CaCl ₂ 2H 0, 0.2 g; Difco Yeast Extract, 2.0 gm; FeSO,, 10 mg; reducing agent (2.5 gm cys- tein HC1 plus 2.5 gm Na _£ S 9H _£ 0 per 200 ml H ₂ 0) 20 ml. K0H was utilized to adjust the pH to 7.2. Where indicated, inorganic py¬ rophosphate (PP.) (filter sterilized) was added giving a final concentration of 0.05% in the medium per liter. The lactate-sul- fate medium contained per liter: sodium lactate (60%), 12.5 ml;

O ΓI

NH ₄ C1, 2.0 g; MgS0 ₄ 7H ₂ 0, 0.2 g; K ₂ HP0 _4> 0.5 g; CaCl ₂ 2H ₂ 0, 0.2 g; Difco Yeast Extract, 1.0 g; Na S 9H ₂ 0, 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 acti¬ vities.

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 anae- robically 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 mi¬ crobial 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 micro¬ organisms. In Table 3, below, a list of microorganisms effected by inorganic pyrophosphate is presented.

Table 3. The Effects of Pyrophosphate on the Growth of Various Microorganisms

Growth Growth Fixed Carbon Electron Support¬ Stimulat¬ Acceptor ing ing (Sink)

Dt. nigri¬ ficans Acetate; Sulfate Yeast Extract Dt. ruminis Acetate; Sulfate Yeast Extract

Dt. orientis Acetate; Sulfate Yeast Extract

Thiobacillus None Nitrate denitrificans ( a ₂ S _£ 0 ₃ ) Methanobac- terium Yeast Extract CO., Casitone

Thermoauto- trophicum Methanosarcina barkerii ? + Acetate; Acetate Yeast Extract

(acetate Casitone methanol)

Clost _. ridium thermocellum + Yeast Extract

Rhodopseudomonas capsulata ? + Acetate acetate

Thermoanaero¬ bacter - ethanolicus + Yeast Extract None

Desulfovibrio vulgaris -

Escherichia coli -

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, Mar¬ seille, France. Cellulose (Avicel) , 5.0 g/1; NaHC0 ₃ , 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; (NH^SO^ 0.9 g/1; KH ₂ P0 ₄ , 0.45 g/1; MgS0 ₄ , 0.09 g/1; CaCl , 0.09 g/1; ₂ HP0 ₄ , 0.45 g/1; Cysteine, 0.5 g/1.) 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

Methant i Formation

(m moles)

Days Minus Pyrophosphate Plus Pyrophosphate

7 0.03 0.06

9 0.04 0.15

11 0.07 0.64

^' 13 0.15 2.04

15 0.24 2.67

17 0.38 3.63

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.

Methane Formation (m moles)

Days Minus Pyrophosphate Plus Pyrophosphate

3 0.18 0.26

5 0.77 1.23

7 1.40 3.92 9 1.63 6.38

11 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. 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 re- o lease of metal ions is followed as a function of time at 30 C. In the presence of inorganic pyrophosphate, there is an increased re- lease 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 forma- o tion 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 growth of the Thiobaci¬ lli 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. KH _£ P0 ₄ , 1.5 g/985 ml glass distilled water; K ₂ HPO ₄ .3H ₂ 0, 2.9 g/985 ml glass distilled water; NH.C1, 1.0 g/985 ml glass dis¬ tilled water; NaCl, 0.9 g/985 ml glass distilled water; MgCl ₂ - 6H 0, 0.2 g/985 ml glass distilled water; CaCl ₂ .2H ₂ 0, 0.05 g/985 glass ml distilled water; NaSeO-, 0.017 mg/985 ml glass distilled water; Mineral Solution, 10 ml/985 ml glass distilled water; Vita¬ min Solution, 5 ml/985 ml glass distilled water; Reazurin (0.2%), 1.0 ml/985 ml glass distilled water.) containing inorganic pyro¬ phosphate. Increased yields of vitamin B._ 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; Casi¬ tone, 2.5 g/1; Cellobiose, 0.2 g/1; NaCl, 0.9 g/1; (NH ₄ ) ₂ S0 ₄ , 0.9 g/1; KH ₂ P0 ₄ , 0.45 g/1; MgS0 ₄ , 0.09 g/1; CaCl ₂ , 0.09 g/1; K ₂ H?0 ₄ , 0.45 g/1; Cysteine, 0.5 g/1.) supplemented with inorganic pyro¬ phosphate. 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 bio- snythesis of the phosphotransferase is transferred to _E. coli us¬ ing techniques known in the art for the transfer of deoxyribonu¬ cleic 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 re¬ plicon 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, uti¬ lization of specific feedstocks, or the like.) allowing the micro¬ organism 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 microor¬ ganisms used in commercial and industrial applications such as leaching of low grade pyrite ores, desulfurization of coal, con¬ version of biomass to ethanol, and conversion of biomass to meth¬ ane.