[go: up one dir, main page]

WO1991013996A1 - Procede pour obtenir une bacterie thermophile anaerobie, bacterie ainsi obtenue et son utilisation pour la fermentation d'hydrates de carbone - Google Patents

Procede pour obtenir une bacterie thermophile anaerobie, bacterie ainsi obtenue et son utilisation pour la fermentation d'hydrates de carbone Download PDF

Info

Publication number
WO1991013996A1
WO1991013996A1 PCT/NL1991/000038 NL9100038W WO9113996A1 WO 1991013996 A1 WO1991013996 A1 WO 1991013996A1 NL 9100038 W NL9100038 W NL 9100038W WO 9113996 A1 WO9113996 A1 WO 9113996A1
Authority
WO
WIPO (PCT)
Prior art keywords
bacterium
fermentation
medium
range
inulin
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/NL1991/000038
Other languages
English (en)
Inventor
Wilhelmus Jurriën DRENT
Jan Cornelis Gottschal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rijksuniversiteit Groningen
Original Assignee
Rijksuniversiteit Groningen
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 Rijksuniversiteit Groningen filed Critical Rijksuniversiteit Groningen
Publication of WO1991013996A1 publication Critical patent/WO1991013996A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium

Definitions

  • thermophilic bac ⁇ terium thus obtainable bacterium and its use for the fermentation of carbohydrates.
  • the invention relates to a method for obtaining an anaerobic bacterium capable of producing succinate.
  • yeast extract is added to the medium.
  • concentration in which it is added is at least 1 g/1.
  • Anaerobic bacteria capable of producing succinate are described in the European patent applic ⁇ ation 0 249 773 « The use of various strains of succinate producing bacteria in the fermentation of carbohydrates such as dextrose, sucrose, fructose, lactose, soluble starches and corn syrups is described.
  • the fermentation is conducted in an aqueous medium containing dissolved carbondioxide and nutrients and growth factors needed for reproduction and growth of the micro-organisms employed.
  • the concentration of carbohydrate in the medium lies between 20 and 100 g/1, as a lower concentr ⁇ ation than 20 g/1 would yield too little succinate for practical recovery.
  • Yeast extract is present at a con ⁇ centration of 10 g/1.
  • the pH-value is maintained between 5.8-6.8. At higher pH-values the main product is lactate rather than succinate.
  • the fermentation process is preferably carried out at a temperature of about 38 * 0.
  • an anaerobic thermophilic bacterium comprising isolation of a carbohydrate fermenting bacterium, which is capable of producing succinate at a temperature within a range of 4 ⁇ -8 ⁇ °C, using carbohydrate as a major source of carbon and energy.
  • bacteria are obtained which may be used for improving fermentation processes, e.g. processes for the production of organic compounds by fermentation of carbohydrates.
  • succinate producing micro-organisms are not interesting from a biotechnological point of view for a number of reasons, e.g. succinic acid is formed in too low concentrations to make the recovery of the ferment- ation product economically feasible and the fermentative bacteria tend to lyse leading to an unstable production process.
  • thermophilic bacteria may offer a number of advantages compared with the production process using mesophilic bacteria amongst others increased productivity as the reaction rate of organisms and enzymes increase and mass cultivation of thermophilic bacteria will be cheaper than that of mesophilic bacteria due to reduced contamination problems (for a complete survey of the advantages for using thermophilic bacteria in biotechnological processes see Sonnleitner, Adv. in Biochem. Eng. 28: 69 ⁇ 138).
  • the isolation method according to the invention it is preferable for the isolation method according to the invention to use a nutrient medium which contains yeast extract in the range of 0-1 wt . % , preferably 0.01-0.1 vt . % , in particular about 0.01 vt . % , based on the total weight of said medium.
  • yeast extract present in the medium in an amount of more than 1 vt . % results in unpractically low numbers of successful isolations of anaerobic thermophilic succinate producing carbohydrate fermenting bacteria.
  • the yeast extract need not be present in the nutrient medium, a small amount thereof (preferably less than 0.1 wt . % ) appears to favour the growth of the bacteria in question.
  • a 90 % success rate of isolation of anaerobic thermophilic succinate producing carbohydrate fermenting bacteria was found when yeast extract was present in a concentration of 0.01 wt . % , based on the total weight of said medium.
  • polysaccharide materials may be used.
  • such polysaccharides are soluble in the liquid nutrient medium which - in general - comprises water.
  • good results were obtained by using dextrans , fructans and xylans as polysaccharides, inulin or starch being examples of preferred materials.
  • the enrichment according to the present invention, for example using inulin can be carried out in a broad pH-range, e.g. between pH 5 and 9 . This is surprising because as yet only a few inulinases with a pH optimum above pH 5 have been found, viz.
  • Panaeolus papillonacens Debaromyces cantarelii and Arthrobacter urafaciens
  • inulinase a non-specific- fruetofuranisodase from the mushroom Panaeolus .
  • Can. Journal of Microbiology 3_3_. 520-52 Guiraud J.P., Bernit, C. Gelzy, P., (1982), Inulinase of Debaromyces cantarellii. Folia Microbiologica 27_, 19" 24; Uchimyama, T., Niwa, S. & Tanaka, K. (1973).
  • Bacteria of the type which may be obtained according to the method of the invention, are present in many natural sources. They were e.g. isolated via en ⁇ richment in batch and continuous cultures, after in- oculation with samples originating from a heat-tower of a sugar factory, soil around a Jerusalem artichoke, fresh cow manure and mud from a tropical pond belonging to the Botanical Garden of the University of Groningen, the Netherlands.
  • the cells of the novel species of Gram- positive, strictly anaerobic, thermophilic bacteria were rod-shaped and non-motile.
  • the G+C content of the DNA was 35«8 +_ 1.0 mol % .
  • Growth on inulin was possible between 40 and 65 * 0, with an optimum around 5 ⁇ * C.
  • the present invention relates to bacterium which may be obtained according to any of the methods of the invention described in the above. It will be clear that for obtaining the bacteria according to the invention further modifications of the conditions may be possible.
  • the present invention relates to bacteria of the species Clostridium thermosuccinogenes .
  • the bacteria according to the invention are capable of fermenting a great number of sugars.
  • the present invention also relates to a method for the fermentation of carbohydrates with a succinate producing bacterium in a medium containing carbohydrates metabol- isable by said bacterium and further nutrients required for growth, wherein said fermentation is carried out at a temperature within a range of 40-80 * 0, preferably by using one of the bacteria indicated in the above.
  • the temper ⁇ ature preferably lies in the range of 50-75'C, corresponding to the optimum growth of the bacteria used.
  • poly- saccharide materials may be used as source of energy and carbon, such as xylan, dextran or fructan, e.g. inulin or starch.
  • xylan e.g. dextran or fructan
  • fructan e.g. inulin or starch.
  • An especially preferred polysaccharide is material from the Jerusalem artichoke. Another useful material may be chicory.
  • thermophilic bacterium a rather low carbo ⁇ hydrate concentration is used in the growth medium during the method for fermentation of carbohydrates a higher carbohydrate concentration is used. It will be self-evident that for economical reasons the highest possible concentration of carbohydrate is preferred.
  • the same pH-values as during the method for isolating the bacteria may be used.
  • a pH- value of about 7 is preferred.
  • the method of the invention is well-suited for the production of succinate from inulin as higher concentrations are obtained upon comparison with the prior art.
  • the present invention also relates to compounds, obtained according to the fermentation processes of the invention. Furthermore, the present invention relates to the use of the specific bacteria in the fermentation industry.
  • the bacteria were enriched from fresh cow manure, beet pulp from the C.S.M. sugar refinery at
  • the bacteria were cultivated at 8 *c fi t a pH of 7 «0 +_ 0.2 using a basal medium containing the following components (g/1): NaCl, 1.2; MgCl 2 .6H 2 0, 0.4; KCl, 0-3;
  • the following stock solutions were sterilised separately and added aseptically to the mineral medium using syringes: NaHCOo (84 g/1), NaH P0
  • the vitamin and the trace elements solution were filter-sterilised, the other components were autoclaved.
  • Enrichments The bacteria were enriched in batch culture, using 600 ml bottles filled with 250 ml bicarbonate- buffered basal medium and 1.5 g/1 inulin as the sole source of carbon and energy. The same medium was used for enrichment in continuous culture. Pure cultures were obtained after diluting in culture tubes with a butyl rubber septum and a screw cap, and subsequently plating the highest dilution with visible growth on agar plates (medium supplemented with 2 % agar) . These plates were in a 2.5 1 jar incubated at 60 * C, to which 0.7 nil 10 mM Na S was added in order to maintain anaerobic conditions. After repeated plating the strains were isolated. Growth experiments
  • ⁇ max The relationship between the ⁇ max and the pH was determined in MES-buffered medium at low pH, and Tris- HC1 at high pH. In the other experiments bicarbonate was used, all substrates were filter-sterilised (0.2 ⁇ m) .
  • Alcohols, short-chain fatty acids, both volatile and non-volatile were analyzed by gas chromatography [Laanbroek, H.J., Geerligs, H.J., Sijtsma, L. & Veldkamp, H., (1984), Competition for sulfate and ethanol among Desulfobacter, Desulfobulbus , and Desulfovibrio species isolated from interdial sediments. Applied Environmetal Microbiology 4j , 329 ⁇ 334], [Nanninga, H.J. & Gottschal, J.C. (1985). Amino acid fermentation and hydrogen transfer in mixed cultures. FEMS Microbiology Ecology, 3_1.
  • Inulinase activity was determined by measuring the appearance of reducing sugars with the method of Somogyi using cell culture, culture supernatant and cells resuspended in 0.3 phosphate buffer after centrifugation (12,000 g ) .
  • the reaction mixture contained 2 ml sodium phosphate buffer (30 mM, pH 6.8), inulin (6 g/1) and 1.8 ml enzyme containing sample.
  • the cell culture was inactivated by flushing the culture with air. The formation of fermentation products could never be measured, during enzyme activity measurements.
  • One unit of inulinase activity is defined as the amount of enzyme catalysing the liberation of 1 ⁇ mol reducing sugars min " l. Specific enzyme activities are expressed per milligram of Cell-carbon.
  • cytochromes The presence of cytochromes was investigated by recording air- oxidized versus dithionite-reduced spectra of whole cells and cell free extracts with an Aminco spectrophotometer type DW2a.
  • the mol % C+G was determined using ultracentrifugation and buoyant density measurements [DSM, Braunschweig].
  • Isolated strains Four different strains were isolated, using different techniques of enrichment and sources for inoculation (Table 1).
  • thermophilic strains were rod shaped, 2 ⁇ m to 4 ⁇ m length and 0.3 ⁇ to 0.4 ⁇ m in width. Frequently spores were observed in stationary cultures. The diameter of the spores was approximately 0.6 ⁇ m. After negative staining with uranyl acetate, it can be seen under the electron microscope that the cells of the type strain IC are peritrichously flagellated. However, no motility was observed in phase contrast microscopy.
  • the inital concentration of the tested substrate is indicated between brackets. All strains used fructose, glucose, galactose, xylose, ribose, sucrose, lactose, maltose, cellobiose, raffinose, H 2 /C0 2 ( 8 ⁇ %/20% ) and starch (monosaccharides , each 10 mM; disaccharide 5 mM; trisaccharide 3 «33 mM and insoluble substrates (1.5 g/1).
  • Figure 2 Kinetics of growth and inulinase activity of strain IC in batch-culture on inulin medium (1.5 g/1).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour obtenir une bactérie thermophile anaérobie consistant à isoler une bactérie de fermentation d'hydrates de carbone capable de produire le succinate à une température comprise entre 40° et 80 °C en utilisant un hydrate de carbone en tant que source principale de carbone et d'énergie. On décrit une bactérie pouvant être obtenue par ledit procédé, ainsi qu'un procédé de fermentation d'hydrates de carbone avec une bactérie produisant le succinate, à une température comprise entre 40° et 80 °C et dans un milieu contenant des hydrates de carbone métabolisables par ladite bactérie.
PCT/NL1991/000038 1990-03-14 1991-03-13 Procede pour obtenir une bacterie thermophile anaerobie, bacterie ainsi obtenue et son utilisation pour la fermentation d'hydrates de carbone Ceased WO1991013996A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US49340890A 1990-03-14 1990-03-14
US493,408 1990-03-14

Publications (1)

Publication Number Publication Date
WO1991013996A1 true WO1991013996A1 (fr) 1991-09-19

Family

ID=23960115

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL1991/000038 Ceased WO1991013996A1 (fr) 1990-03-14 1991-03-13 Procede pour obtenir une bacterie thermophile anaerobie, bacterie ainsi obtenue et son utilisation pour la fermentation d'hydrates de carbone

Country Status (2)

Country Link
AU (1) AU7490891A (fr)
WO (1) WO1991013996A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114196588A (zh) * 2021-12-10 2022-03-18 中国科学院青岛生物能源与过程研究所 一种嗜热厌氧琥珀酸梭菌株及其利用木质纤维素产琥珀酸的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0249773A2 (fr) * 1986-06-11 1987-12-23 Michigan Biotechnology Institute Procédé de production d'acide succinique par fermentation anaérobie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0249773A2 (fr) * 1986-06-11 1987-12-23 Michigan Biotechnology Institute Procédé de production d'acide succinique par fermentation anaérobie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 57, No. 2, February 1991, Washington D.C., W.J. DRENT et al., "Fermentation of Inulin by Clostridium Thermosuccinogenes sp.nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats", pages 455-462. *
BIOTECHNOLOGY LETTERS, Vol. 7, No. 11, November 1985, Kew, England, B. OLLIVIER et al., "Thermophilic Methanogenesis from Sugar Beet Pulp by a Defined Mixed Bacterial Culture", pages 847-852. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114196588A (zh) * 2021-12-10 2022-03-18 中国科学院青岛生物能源与过程研究所 一种嗜热厌氧琥珀酸梭菌株及其利用木质纤维素产琥珀酸的方法
CN114196588B (zh) * 2021-12-10 2023-06-09 中国科学院青岛生物能源与过程研究所 一种嗜热厌氧琥珀酸梭菌株及其利用木质纤维素产琥珀酸的方法

Also Published As

Publication number Publication date
AU7490891A (en) 1991-10-10

Similar Documents

Publication Publication Date Title
Giraud et al. Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum
Giallo et al. Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10
US5350692A (en) Microorganisms useful for hydrogen gas production
Maroune et al. Interactions between rumen amylolytic and lactate‐utilizing bacteria in growth on starch
Sunnotel et al. Pectinolytic activity of bacteria isolated from soil and two fungal strains during submerged fermentation
EP1583838B1 (fr) Preparation d'acide lactique d'un substrat contenant des pentoses
JPWO2012008585A1 (ja) 新規乳酸菌及びそれを用いたｌ−乳酸の生産方法
Ishizaki et al. L-Lactate production from xylose employing Lactococcus lactis IO-1
US20130143288A1 (en) Isolated bacteria, methods for use, and methods for isolation
Selvakumar et al. Comparative studies on inulinase synthesis by Staphylococcus sp. and Kluyveromyces marxianus in submerged culture
Cortezi et al. Temperature effect on dextransucrase production by Leuconostoc mesenteroides FT 045 B isolated from Alcohol and Sugar Mill Plant
Vandamme et al. Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations
Marwoto et al. Enhancement of (R, R)-2, 3-butanediol production from xylose by Paenibacillus polymyxa at elevated temperatures
GOYAL et al. Regulation of dextransucrase productivity of Leuconostoc mesenteroides NRRL B-512F by the maintenance media
Parekh et al. High concentrations of acetate with a mutant strain of C. thermoaceticum
CN107686822A (zh) 一种发酵产低色素黄原胶的方法
US7083955B2 (en) Preparation of lactic acid from a pentose-containing substrate
Kannan et al. Ethanol fermentation characteristics of Thermoanaerobacter ethanolicus
CN100451106C (zh) 一株产结冷胶的少动鞘氨醇单孢菌及其应用
Emtiazi et al. A novel nitrogen-fixing cellulytic bacterium associated with root of corn is a candidate for production of single cell protein
Pasti et al. The effects of various nutrients on extracellular peroxidases and acid-precipitable polymeric lignin production by Streptomyces chromofuscus A2 and S. viridosporus T7A
Rode et al. Adaptation of Rhodopseudomonas sphaeroides to Growth on d-(—)-Tartrate and Large-Scale Production of a Constitutive d-(—)-Tartrate Dehydratase During Growth on dl-Malate
Drent et al. Fermentation of inulin by a new strain of Clostridium thermoautotrophicum isolated from dahlia tubers
WO1991013996A1 (fr) Procede pour obtenir une bacterie thermophile anaerobie, bacterie ainsi obtenue et son utilisation pour la fermentation d'hydrates de carbone
RU2751490C1 (ru) Способ получения продуктов сбраживания глюкозы из целлюлозосодержащего сырья и целлюлозосодержащих отходов термофильной бактерией

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA