WO1983002952A1 - Stimulation de croissance bacterienne par du pyrophosphate inorganique - Google Patents

Stimulation de croissance bacterienne par du pyrophosphate inorganique Download PDF

Info

Publication number
WO1983002952A1
WO1983002952A1 PCT/US1983/000227 US8300227W WO8302952A1 WO 1983002952 A1 WO1983002952 A1 WO 1983002952A1 US 8300227 W US8300227 W US 8300227W WO 8302952 A1 WO8302952 A1 WO 8302952A1
Authority
WO
WIPO (PCT)
Prior art keywords
microorganisms
process according
inorganic pyrophosphate
growth
pyrophosphate
Prior art date
Application number
PCT/US1983/000227
Other languages
English (en)
Inventor
Jr. Harry D. Peck
Nancy K. Hart
Chi-Li Liu
Jean Legall
Original Assignee
University Of Georgia Research Foundation, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Georgia Research Foundation, Inc. filed Critical University Of Georgia Research Foundation, Inc.
Priority to BR8305196A priority Critical patent/BR8305196A/pt
Publication of WO1983002952A1 publication Critical patent/WO1983002952A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/023—Methane
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C22—METALLURGY; FERROUS OR NON-FERROUS ALLOYS; TREATMENT OF ALLOYS OR NON-FERROUS METALS
    • C22B—PRODUCTION AND REFINING OF METALS; PRETREATMENT OF RAW MATERIALS
    • C22B3/00—Extraction of metal compounds from ores or concentrates by wet processes
    • C22B3/18—Extraction of metal compounds from ores or concentrates by wet processes with the aid of microorganisms or enzymes, e.g. bacteria or algae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P10/00—Technologies related to metal processing
    • Y02P10/20—Recycling

Definitions

  • 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.
  • 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.
  • ATP adenosine triphosphate
  • 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
  • respiratory sulfate reduction is hydrolyzed to orthophosphate (P i ) by inorganic pyrophosphatase (Eq. 2).
  • Adenosine triphosphate can then be produced from acetyl phosphate and adenosine diphosphate (ADP) by acetate kinase (Eq. 4).
  • 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.
  • 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.
  • 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.
  • 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..
  • Desulfotomaculum nigrificans, Desulfotomaculum ruminis, and Desulfotomaculum orientis were grown on a medium containing inorganic pyrophosphate, acetate, yeast extract, sulfate, and salts.
  • 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.
  • crude enrichment cultures from a marine spartina marsh and fresh water marshes were similarly grown using inorganic pyrophosphate as an energy source.
  • Example I describes conditions for the anaerobic growth of Desulfotomaculum nigrificans on inorganic pyrophosphate.
  • Table I compares growth on various media.
  • the basal medium containing acetate, yeast extract, sulfate, and salts does not support growth of the microorganism.
  • the basal medium was supplemented with inorganic pyrophosphate, growth was better than obtained under usual growth conditions with lactate plus sulfate.
  • 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.
  • 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.
  • Lactate-Sulfate Medium 0.505 *Measured at 580 nm; average of duplicate flasks after forty eight hours incubation at 55oC under argon.
  • Example II 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.
  • Reaction mixtures contained 5.0 mM BV 2+ , 10.0 mM dithiothreitol, 100 mM potassium phosphate buffer (pH 7.4), and 20 mM sodium formate for the formate BV 2+ reaction and 5.0 mM BV 2+ , 2.0 mM reduced coenzyme A, 100 mM potassium phosphate (pH 7.4), and 20.0 mM sodium pyruvate for the pyruvate-BV 2+ reaction. Both assays were performed under argon in Thunberg covettes.
  • c-type cytochromes were determined by difference spectroscopy at 538 to 551 nm (40).
  • 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.
  • Adenosine triphosphate sulfurylase was determined with MoO 4 2- as described by Wilson and Bandurski (Wilson, L. G. and R.
  • 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;
  • 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 crochet 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 barkieri 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.
  • 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.
  • the yield of vitamin B 12 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.
  • 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.
  • 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; Na 2 SO 4 , 0.4 gm; MgSO 4 7H 2 O, 0.2 g; MgCl 2 6H 2 O, 1.8 gm; K 2 HPO 4 , 0.5 g; CaCl 2 2H 2 0, 0.2 g; Difco Yeast Extract, 2.0 gm; FeSO 4 , 10 mg; reducing agent (2.5 gm cystein HCI plus 2.5 gm Na 2 S 9H 2 O per 200 ml H 2 O) 20 ml. KOH was utilized to adjust the pH to 7.2.
  • 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; NH 4 C1, 2.0 g; MgSO 4 7H 2 O, 0.2 g; K 2 HPO 4 , 0.5 g; CaCl 2 2H 2 O, 0.2 g; Difco Yeast Extract, 1.0 g; Na 2 S 9H 2 O, 0.25 g.
  • Table 1 shows comparative growth after forty-eight hours.
  • 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.
  • 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.
  • 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.
  • inorganic pyrophosphate 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.
  • Table 3 below, a list of microorganisms effected by inorganic pyrophosphate is presented.
  • 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; NaHCO 3 , 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; (NH 4 ) 2 SO 4 , 0.9 g/l; KH 2 PO 4 , 0.45 g/l; MgSO 4 , 0.09 g/l; CaCl 2 , 0.09 g/l; K 2 HPO 4 , 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.
  • 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.
  • 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.
  • a fermentative anaerobic bacterium for example, Thermoanaerobacter ethanolicus with and without inorganic pyrophosphate and inoculated at the optimal growth temperature for the bacterium.
  • inorganic pyrophosphate the fermentation products are altered such that a higher concentration of the desired product is obtained.
  • 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.
  • 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.
  • Methanosarcina barkieri is inoculated into a standard medium (Weiner, P. J. and Zeikus, J. G. , Arch. Microbiol. 119:46-57, 1978.
  • KH 2 PO 4 1.5 g/985 ml glass distilled water
  • K 2 HPO 4 .3H 2 O 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
  • MgCl 2 MgCl 2 .
  • 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; (NH 4 ) 2 SO 4 , 0.9 g/1; KH 2 PO 4 , 0.45 g/1; MgSO 4 , 0.09 g/1; CaCl 2 , 0.09 g/1; K 2 HPO 4 , 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.
  • 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.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Mechanical Engineering (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Metallurgy (AREA)
  • Environmental & Geological Engineering (AREA)
  • Materials Engineering (AREA)
  • Manufacturing & Machinery (AREA)
  • Geology (AREA)
  • Geochemistry & Mineralogy (AREA)
  • General Life Sciences & Earth Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédé de croissance de microorganismes dans lequel on utilise du pyrophosphate inorganique en tant que source d'énergie pour produire de l'adénosine triphosphate. Des microorganismes avec les enzymes acétate phosphotransférase et acétate kinase sont utilisés dans un milieu contenant une source de carbone fixé additionnée de phyrophosphate inorganique. Ce procédé peut être utilisé pour résoudre le problème posé par la croissance lente ou faible de microorganismes utilisés dans des procédés industriels tels que lessivage de minerai de pyrite de grade inférieur, désulfuration du charbon, conversion de biomasse ou de cellulose en méthanol, et conversion de biomasse ou de cellulose en éthanol.
PCT/US1983/000227 1982-02-26 1983-02-22 Stimulation de croissance bacterienne par du pyrophosphate inorganique WO1983002952A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
BR8305196A BR8305196A (pt) 1982-02-26 1983-02-22 Processos para crescimento de microorganismos e para gerar adenosina tritosfato

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35274282A 1982-02-26 1982-02-26
US352,742 1982-02-26

Publications (1)

Publication Number Publication Date
WO1983002952A1 true WO1983002952A1 (fr) 1983-09-01

Family

ID=23386302

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1983/000227 WO1983002952A1 (fr) 1982-02-26 1983-02-22 Stimulation de croissance bacterienne par du pyrophosphate inorganique

Country Status (3)

Country Link
EP (1) EP0101721A4 (fr)
JP (1) JPS6027511B2 (fr)
WO (1) WO1983002952A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0181769A1 (fr) * 1984-11-08 1986-05-21 International Technology Corporation Stimulation des bactéries aérobiques
US5362639A (en) * 1991-01-10 1994-11-08 Monsanto Company Method to increase anaerobic fermentation rates
WO2009067471A2 (fr) * 2007-11-19 2009-05-28 Novozymes A/S Procédés de fabrication de produits de fermentation
US20120064609A1 (en) * 2010-09-10 2012-03-15 Synthetic Genomics, Inc. Solubilization of coal or lignocellulose biomass
US20130164732A1 (en) * 2011-12-09 2013-06-27 The Curators Of The University Of Missouri Inorganic pyrophosphate and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62152203A (ja) * 1985-12-26 1987-07-07 Yuken Kogyo Kk 比例電磁弁駆動用パワ−増幅器

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3105014A (en) * 1961-12-07 1963-09-24 Tidewater Oil Company Bacterial treatment of media containing hydrocarbons and sulfides
US4237224A (en) * 1974-11-04 1980-12-02 Board Of Trustees Of The Leland Stanford Jr. University Process for producing biologically functional molecular chimeras

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3105014A (en) * 1961-12-07 1963-09-24 Tidewater Oil Company Bacterial treatment of media containing hydrocarbons and sulfides
US4237224A (en) * 1974-11-04 1980-12-02 Board Of Trustees Of The Leland Stanford Jr. University Process for producing biologically functional molecular chimeras

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Federation Proceedings, Vol. 36 issued 1977, "Some Reactions in Which Inorganic Pyrophosphate Replaces ATP and Serves as a Souree of Energy" H.C. WOOD, see p 2197/2205 *
Federation Proceedings, Vol.40, issued 1981, "The Utilization of Inorganic Polyphosphate by Propionic Acid Bacteria and Polyphosphate Glucokinase." N.H. GOSS et al, see page 1867 *
See also references of EP0101721A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0181769A1 (fr) * 1984-11-08 1986-05-21 International Technology Corporation Stimulation des bactéries aérobiques
US4727031A (en) * 1984-11-08 1988-02-23 International Technology Corporation Nutrient for stimulating aerobic bacteria
US5362639A (en) * 1991-01-10 1994-11-08 Monsanto Company Method to increase anaerobic fermentation rates
WO2009067471A2 (fr) * 2007-11-19 2009-05-28 Novozymes A/S Procédés de fabrication de produits de fermentation
WO2009067471A3 (fr) * 2007-11-19 2009-07-09 Novozymes As Procédés de fabrication de produits de fermentation
US8227221B2 (en) 2007-11-19 2012-07-24 Novozymes North America, Inc. Processes of producing fermentation products
US20120064609A1 (en) * 2010-09-10 2012-03-15 Synthetic Genomics, Inc. Solubilization of coal or lignocellulose biomass
US9528061B2 (en) 2010-09-10 2016-12-27 Synthetic Genomics Inc. Solubilization of coal or lignocellulose biomass
US20130164732A1 (en) * 2011-12-09 2013-06-27 The Curators Of The University Of Missouri Inorganic pyrophosphate and uses thereof
US8771934B2 (en) * 2011-12-09 2014-07-08 The Curators Of The University Of Missouri Inorganic pyrophosphate and uses thereof

Also Published As

Publication number Publication date
EP0101721A1 (fr) 1984-03-07
JPS6027511B2 (ja) 1985-06-29
EP0101721A4 (fr) 1986-04-15
JPS59500253A (ja) 1984-02-23

Similar Documents

Publication Publication Date Title
Katsyv et al. Overcoming energetic barriers in acetogenic C1 conversion
Lynd et al. Carbon monoxide metabolism of the methylotrophic acidogen Butyribacterium methylotrophicum
Ben-Bassat et al. Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii
Kampen Nutritional requirements in fermentation processes
Kanai et al. Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1
Nandi et al. Microbial production of hydrogen: an overview
Frankena et al. A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease in Bacillus licheniformis
US7432091B2 (en) Highly efficient hydrogen production method using microorganism
Bibra et al. Single pot bioconversion of prairie cordgrass into biohydrogen by thermophiles
CN104651287A (zh) 一种用于合成甘油葡糖苷的工程菌和应用
Thapa et al. Metabolic engineering of Enterobacter aerogenes to improve the production of 2, 3-butanediol
Chang et al. Formulation of defined media for carbon monoxide fermentation by Eubacterium limosum KIST612 and the growth characteristics of the bacterium
Loubière et al. Kinetics and physiological implications of the growth behaviour of Eubacterium limosum on glucose/methanol mixtures
Fathepure Factors affecting the methanogenic activity of Methanothrix soehngenii VNBF
Archer et al. The DNA sequence and minimal replicon of the Corynebacterium glutamicum plasmid pSR1: evidence of a common ancestry with plasmids from C. diphtheriae
WO1983002952A1 (fr) Stimulation de croissance bacterienne par du pyrophosphate inorganique
Escalante-Semerena et al. Formaldehyde oxidation and methanogenesis
Toda Induction and repression of enzymes in microbial culture
Bryson et al. Energy-dependent transport of nickel by Clostridium pasteurianum
Jeon et al. Activation of ethanol production by combination of recombinant Ralstonia eutropha and electrochemical reducing power
CA1207258A (fr) Stimulation de la multiplication des bacteries par un pyrophosphate inorganique
CN118006698A (zh) 通过生产蚁酸(甲酸)储存气态氢的方法
Hill et al. Bioenergetics and end‐product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation
Kovacs et al. Improvement of biohydrogen production and intensification of biogas formation
Malashenko et al. Role of monooxygenase reaction during assimilation of non-growth substrates by methanotrophs

Legal Events

Date Code Title Description
AK Designated states

Designated state(s): BR JP

AL Designated countries for regional patents

Designated state(s): DE FR GB SE

WWE Wipo information: entry into national phase

Ref document number: 1983901047

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1983901047

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1983901047

Country of ref document: EP