JP5214272B2 - New microorganism - Google Patents

Description

  The present invention relates to a novel microorganism, and more particularly to a new kind of microorganism having the ability to produce ethanol using carbon monoxide or carbon dioxide and hydrogen as substrates.

  Research on new energy is being promoted to combat global warming and reduce consumption of fossil fuels.

  Useful materials such as acetic acid and ethanol are fermented by microorganisms using gaseous carbon dioxide and carbon monoxide, etc. contained in the gas discharged from the gasification and reforming of industrial waste gas and woody biomass. There is a technology to recover.

In particular, in recent years, demand for fuel has been increasing, and ethanol has a high value as a useful product. Furthermore, since carbon dioxide is one of the greenhouse gases, if bacteria that can use carbon dioxide as a carbon source are obtained, the effect of reducing the greenhouse gases can be expected.
Japanese Patent Application Laid-Open No. 1-98472 Acetobacterium genus, Clostridium genus U.S. Pat. No. 5,173,429 Genus Clostridium Japanese translation of PCT publication No. 2002-543793 Publication No. Clostridium genus JP, 2003-339371, A Clostridium genus or its derivative genus

  As microorganisms that produce useful substances such as acetic acid and ethanol using carbon dioxide and carbon monoxide as substrates, only the genus Clostridium has been known (Patent Documents 1 to 4).

  The present inventors have repeated the isolation and screening of microorganisms, selected microorganisms having the ability to produce ethanol using carbon dioxide and carbon monoxide as a substrate, and completed the present invention.

  That is, the subject of this invention is providing the novel microorganisms which have the capability to produce | generate ethanol.

  The other subject of this invention becomes clear by the following description.

  The above problems are solved by the following inventions.

(Claim 1)
Introducing carbon monoxide or synthesis gas consisting of carbon dioxide and hydrogen as a substrate, and having the ability to produce ethanol in an anaerobic environment , Veillonella sp. A novel microorganism deposited as a strain M9 (deposit number NITE P-472) at the Patent Microorganism Depositary, National Institute of Technology and Evaluation .

  ADVANTAGE OF THE INVENTION According to this invention, the novel microorganisms which have ethanol production ability can be provided.

  Embodiments of the present invention will be described below.

  The novel microorganism belonging to the genus Veillonella of the present invention is hereinafter referred to as “the present strain” as necessary.

  This strain is a strain isolated from the methane fermentation treatment fraction of Fukuoka City Sewage Treatment Plant, and has the ability to produce ethanol in addition to acetic acid using carbon dioxide or carbon monoxide as a substrate. It is a new species to which it belongs.

  This strain is Veillonella sp. It has been deposited as Strain M9 (deposit number NITE P-472) at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, and has the following properties. In addition, + shows positive or existence,-shows negative or nothing.

A. Morphological properties (1) Cell shape and size: about 0.3 μm of cocci
(3) Spore presence:

B. Culture properties (1) Clostridium ljungdahlii (CL) medium agar plate culture (* 1)
I) Color: white ii) Surface shape: smooth iii) Transparency: translucent iv) Change in colony morphology due to mutation: −
v) Changes in colony morphology due to culture conditions and physiological conditions:-
* 1 C.I. The medium composition of the ljungdahlii medium is shown below.
NH ₄ Cl 1.00g
KCl 0.10g
MgSO ₄ 0.20 g
NaCl 0.80g
KH ₂ PO ₄ 0.10 g
CaCl ₂ 0.02 g
Na ₂ WO ₄ 0.20 mg
Yeast Extract 1.00g
NaHCO ₃ 1.00 g
Fructose 5.00g
Cysteine-HCl 0.30 g
Na ₂ S 0.30 g
Trace element solution (I) 10ml
Vitamin solution (II) 10ml
Distilled water 1000ml
Agar 15g
pH 5.9
The above (I) Trace element solution and (II) Vitamin solution have the following compositions.
(I) Trace element solution
Nitritriacetic acid 1.5g
MgSO ₄ 3.0 g
MnSO ₄ 0.5g
NaCl 1.0g
FeSO ₄ 0.1g
CoSO ₄ 0.18 g
CaCl ₂ 0.1 g
ZnSO ₄ 0.18 g
CuSO ₄ 0.01g
KAl (SO ₄ ) ₂ 0.02 g
H ₃ BO ₃ 0.01 g
Na ₂ MoO ₄ 0.01 g
NiCl ₂ 0.025g
Na ₂ SeO ₃ 0.3 mg
Distilled water 1000ml
(II) Vitamin solution
Biotin 2.0mg
Folic asid 2.0mg
Pyridoxine-HCl 10mg
Thiamine-HCl 5.0mg
Riboflavin 5.0mg
Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
Vitamin B ₁₂ 0.1mg
P-Aminobenzoic acid 5.0mg
Lipic acid 5.0mg
Distilled water 1000ml
(2) Gelatin puncture culture i) Gelatin liquefaction:-
(3) Litmus milk i) Reaction: Litmus reduction ii) Coagulation: +
(4) B. C. P. Milk i) Reaction: Alkaline

C. Physiological properties (1) Gram staining:-
(2) Reduction of nitrate: +
(3) Production of indole:-
(4) Production of hydrogen sulfide:-
(5) Starch hydrolysis:-
(6) Urease:-
(7) Catalase:-
(8) Range of growth i) Optimum pH: 7.2
ii) Temperature: good growth at 20-42 ° C. (9) Attitude toward oxygen: obligate anaerobic (10) OF test: oxidized form (11) Acid and gas production from sugars i) L-arabinose: Acid (-) / Gas (-)
ii) D-xylose: acid (-) / gas (-)
iii) Glucose: acid (+) / gas (−)
iv) D-mannose: acid (−) / gas (−)
v) Fructose: acid (+) / gas (-)
vi) Maltose: acid (-) / gas (-)
vii) Lactose: acid (-) / gas (-)
viii) D-trehalose: acid (-) / gas (-)
ix) D-sorbitol: acid (-) / gas (-)
x) D-mannitol: acid (-) / gas (-)
xi) Glycerin: acid (-) / gas (-)
xii) D-cellobiose: acid (-) / gas (-)
xiii) esculin: acid (-) / gas (-)
xiv) Salicin: acid (−) / gas (−)
xv) D-meletitol: acid (-) / gas (-)
xvi) D-raffinose: acid (-) / gas (-)
xvii) L-rhamnose: acid (+) / gas (-)

D. Enzymatic reaction i) Arginine dihydrolase: +
ii) α-galactosidase: −
iii) β-galactosidase:-
iv) β-galactosidase-6-phosphate: −
v) α-glucosidase: −
vi) β-glucosidase: −
vii) α-arabinosidase:-
viii) N-acetyl-β-glucosaminidase:-
ix) glutamate decarboxylase:-
x) α-Fucosidase: −
xi) alkaline phosphatase:-
xii) Arginine allylamidase:-
xiii) p-proline allylamidase:-
xiv) Leucylglycine allylamidase:-
xv) Phenylalanine allylamidase:-
xvi) Leucine allylamidase:-
xvii) pyroglutamate allylamidase:-
xviii) tyrosine allylamidase:-
xix) Alanine allylamidase:-
xx) Glycine allylamidase:-
xxi) Histidine allylamidase:-
xxii) Glutamylglutamate allylamidase:-
xxiii) Serine allylamidase:-

E. Molecular phylogenetic analysis based on the base sequence of 16S rDNA The base sequence of 16S rDNA was determined, the DNA database (DDBJ) was accessed, and the homology search of the base sequence of 16S rDNA was performed using the BRAST program. The homology of 16S rDNA with the genus bacteria was less than 97%.

F. Results of Classification / Identification Based on the taxonomic characteristics of this strain by the phenotypic traits, Bergey's Manual of Systematic Bacteriology, Vol. 1, N. R. Krieg, J .; G. Holt (ed), Williams & Wilkins, Baltimore (1984) and Bergey's Manual of Determination Bacteriology (9th ed.), J. Am. G. Holt, N .; R. Krieg, P.A. H. A. Sneath, J .; T.A. Staley, S.M. T.A. As a result of classification and identification with reference to Williams (ed), Williams & Wilkins, Baltimore (1994), this strain was identified as the genus Veillonella.

  In the genus Veillonella, the reference species V. In addition to parvula, V. atypica, V.M. disapar, V.D. criceti, V.M. ratti and the like are known. Known genus Veillonella is V. Only criceti has the ability to ferment fructose, but generally only has the ability to ferment organic acids such as pyruvic acid and lactic acid without fermenting sugars.

  On the other hand, this strain can be grown on fructose (fermentable) and is taxonomically different from other Veillonella spp. In that it also has the ability to ferment glucose.

  In general, molecular phylogenetic analysis based on the base sequence of 16S rDNA can be regarded as homologous if homology is 95% or higher, similar if 97% or higher, and homologous if 99% or higher. It is said that.

  The 16S rDNA result of this strain was less than 97% homologous to other Veillonella bacteria, indicating that it was a different species from the known Veillonella strains.

  This strain is characterized by producing ethanol in addition to acetic acid from glucose and fructose, and also producing ethanol in addition to acetic acid using carbon monoxide or carbon dioxide and hydrogen as substrates.

  The method for producing ethanol using this strain is not particularly limited.

  Examples of a method for producing ethanol using carbon monoxide or carbon dioxide and hydrogen as substrates include a method in which an anaerobic state is performed by supplying a microorganism with a synthesis gas composed of carbon monoxide or carbon dioxide and hydrogen.

  The ethanol production mechanism is as follows.

  FIG. 1 is a schematic diagram of an ethanol production pathway that is considered to be included in this strain.

  Many microorganisms with this pathway only produce acetic acid or organic matter that forms cells from acetyl CoA, but this strain can also produce ethanol in addition to acetic acid.

  Examples of the present invention will be described below, but the present invention is not limited to such examples.

Example 1
Ethanol production ability by this strain was confirmed.

First, 20 ml of sterilized CL medium inoculated with this strain was placed in a 50 ml pressure-resistant vial, CO / CO ₂ gas (CO: CO ₂ = 8: 2) was sealed, and the mixture was shaken at 150 rpm for 24 to 48 hours at 37 ° C. Culture was performed to prepare a culture solution.

The culture solution was transferred to the experimental apparatus shown in FIG. 2 (fermentor effective volume 1 L) while being pressurized, and CO / CO ₂ gas was re-encapsulated. An ethanol production experiment was conducted for 3 days under conditions of pH 7.0, temperature 37 ° C., stirring 100 rpm, and CO / CO ₂ gas circulation 0.1 vvm.

  It was possible to obtain 0.22 g of ethanol per 1 g of microbial cells per day.

Schematic diagram of ethanol production pathway The figure which shows the apparatus which performs ethanol production used for the Example

Claims (1)

Introducing carbon monoxide or synthesis gas consisting of carbon dioxide and hydrogen as a substrate, and having the ability to produce ethanol in an anaerobic environment , Veillonella sp. A novel microorganism deposited as a strain M9 (deposit number NITE P-472) at the Patent Microorganism Depositary, National Institute of Technology and Evaluation .

Images