JPH0759187B2 - Bacteria suitable for the production of ethanol from molasses - Google Patents

Description

Description: BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a bacterium that can be used for producing alcohol for fuel from molasses.

Conventional technology Yeast has been used for industrial ethanol production from sugars, but in recent years, Zymomonas has been used.
Ethanol fermentation by bacteria such as these has begun to attract attention.

Zymomonas bacterium, like yeast, produces about 2 mol of ethanol from 1 mol of glucose, but the productivity of ethanol is much higher than that of yeast, and the fermentation rate is also higher than that of yeast. Fuel alcohol production from biomass is of great interest. But,
Zymomonas bacteria are also inferior to yeast in that fermentable sugars are limited to glucose, fructose and sucrose.

In addition, as sugar raw materials used in the production of alcohol for fuel by the fermentation method, cane juice, molasses, saccharified liquid of grain, etc. are generally used, but molasses, which is a typical sugar raw material in Japan, has a high salt concentration. Since it is high, the amount of ethanol produced from molasses is low in the existing Zymomonas bacteria having weak salt tolerance.

Therefore, in order to industrially commercialize the production of alcohol for fuel from molasses using Zymomonas bacteria, it is considered necessary to obtain a strain having properties that surpass existing Zymomonas bacteria in terms of salt resistance and the like. .

[Outline of Invention]

An object of the present invention is to provide a novel microbial strain suitable for the production of alcohol for fuel from molasses, which has properties that surpass existing Zymomonas bacteria in terms of salt tolerance and the like.

The present inventor has eagerly searched for a microorganism that surpasses existing Zymomonas bacteria in terms of salt tolerance, fermentation temperature, sugar utilization and the like in nature. As a result, they have found that the bacteria isolated from sugarcane juice collected in Brazil are superior in salt resistance to existing Zymomonas bacteria and that the amount of ethanol produced from molasses is improved, and the present invention was completed.

SUMMARY Zymomonas mobilis according to the present invention contains glucose 5.
In a liquid medium (pH 6.5) consisting of 0 (w / v)%, monopotassium phosphate 0.2 (w / v)%, yeast extract 1.0 (w / v)% and potassium chloride 2.0 (w / v)%, Initial bacterial count 30 at the level of ^{5 5} cells / ml
At least 1.75 (w / v
(Culture fluid)% ethanol is produced.

Specific examples of such bacteria include Zymomonas mobilis
Subspecies Mobilis B53
No.) and Zymomonas mobilis subspecies mobilis B69 (Ministry of Microbiology Research Institute No. 8923).

Effect The bacterium of the present invention has excellent salt tolerance and a KC1 concentration of 2.0 (w / w
v)% ethanol production capacity in the above medium is at least 1.75 (w / v culture)%, eg at least 1.9
0% (B53 strain) or at least 1.75% (B69 strain).

Considering that the conventional Zymomonas bacterium has poor salt tolerance (see Experimental Examples described below), it should be said that the strong salt tolerance of the bacterium of the present invention was unexpected.

[Specific Description of the Invention]

Microbial collection site The B53 strain and the B69 strain, which are specific examples of the bacterium of the present invention, were isolated by the present inventor from sugar cane juice collected in Brazil.

Deposit numbers B53 and B69 strains have been deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry, and have the following deposit numbers. Strain Accession No. B53 Microindustrial Research Institute No. 8922 B69 Microindustrial Research Institute No. 8923 Mycological properties The main mycological properties of the B53 and B69 strains are as follows.

1) Morphological properties (incubated in standard agar medium ^* at 30 ° C for 24 hours) (1) Bacterial form: rod (2) Size: 1.6-8.0 μm x 1.2-1.6 μm (3) Polymorphism: None (4 ) Motility: None (5) Spore: None (6) Gram stainability: Negative (7) Culture findings: Does not grow in broth medium. Standard medium
Good growth in ^** (standard medium), smooth surface in agar plating, and gloss in all edges. The color is white to cream. Gelatin liquefaction capacity is negative.

* Standard agar medium D-glucose 20g / liter yeast extract 10g / liter agar 20g / liter ** Standard medium D-glucose 20g / liter yeast extract 10g / liter 2) Physiological properties (1) Reduction of nitrate: Not observed (2) Denitrification reaction: Not observed (3) MR test: Negative (4) VP test: Positive (5) Indole production: Not observed (6) Hydrogen sulfide production: Not observed (7) ) Starch hydrolysis: Not allowed (8) Use of citric acid: Not allowed (9) Use of inorganic nitrogen: Available in the form of ammonium salt (10) Dye formation: Not allowed (11) Urease : Negative (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range pH: Growth pH 4.0-8.5 Optimum pH 5.5-7.0 Temperature: Growth temperature 15 ℃ -40 ℃ Optimum temperature 30 ℃ -35 ℃ ( 15) Against oxygen That attitude: facultative anaerobic (16) OF test: Fermentative (Fermentative) (17) Generation of acid and gas from sugars: D-glucose, D- fructose, produces an acid and gas from sucrose. No acid or gas is produced from L-arabinose, D-xylose, D-mannose, D-galactose, maltose, lactose, trehalose, D-sorbit, D-mannitol, inosit, glycerin or starch.

(18) Decomposition products of sugars: About 1 mol of glucose or fructose produces about 2 mol of ethanol and carbon dioxide gas.

(19) Nutritional requirements: Pantothenic acid From the above-mentioned mycological properties, "Virgies Manual of Systematic Bacteriology" Vol. 1 (19
84) (Bergey's Manual of Systematic Bacteriology,
vol.1 (1984) and "International Journal of Systematic Bacteriology" 26th
Volume, pp. 146-157 (1976) (Int.J.Syst.Bacteriol., Vol.
26, p146-157 (1976)).
Mobilis Subspecies Mobilis (Zymomonas mo
bilis subsp.mobilis). However, this bacterium and the known Zymomonas mobilis subspecies mobilis differ in the plasmids they possess in view of their electrophoretic patterns, and also in their salt tolerance, as shown in FIGS. 1-2 and Tables 3-4. Therefore, this bacterium was named Zymomonas mobilis subspecies mobilis B53 and B69.

Culturing The bacterium of the present invention can be grown by culturing in any purposeful medium and culturing conditions to obtain the required amount of cells.

One of the specific examples of the medium and the culture conditions is as shown in the experimental example described later.

Production of ethanol As described above, it is known that ethanol is produced by causing Zymomonas bacteria to act on molasses.
The production of ethanol from molasses by the bacterium of the present invention is essentially the same as the conventionally known method except that the bacterium used is the salt-tolerant bacterium of the present invention.

The molasses targeted by the bacterium of the present invention is typically mother liquor obtained after sufficient recovery of sucrose from sucrose juice until crystallization thereof does not occur, that is, molasses, and such molasses is also typical. Is also suitable in that the effect of the bacterium of the present invention can be maximized because the salt is concentrated. However, molasses targeted by the bacterium of the present invention is not limited to waste molasses as described above, and if necessary, sucrose juice concentrated mother liquor containing sucrose to such an extent that crystallization of sucrose is still possible. It also includes a crude sugar concentrated mother liquor, and a corresponding molasses or concentrated mother liquor for sugar beet sugar. The salt content of the above-mentioned typical molasses, sucrose squeezed waste molasses, is, for example, “Alcohol Handbook” written by Motoki Honda (Fermentation Association).
1963).

Experimental Example Example A small amount of sugarcane juice collected in Brazil (State of Sao Paulo) was inoculated into a medium for accumulation having the following composition.

Media composition for accumulation Yeast extract [Oxoid] 1.0% (w / v) KH ₂ PO ₄ 0.2% (〃) Glucose 2.5% (〃) Fructose 2.5% (〃) Ethanol 5.0 (v / v)% Cabicidin (Kabicidin) ) 100ppm (w / v) Erythromycin 1ppm (〃) pH6.2 Then, this is cultivated at 30 ℃ for 2 to 3 days. It was applied to a plate medium.

Separation agar plate medium Yeast extract [Oxoid] 1.0% (w / v) KH ₂ PO ₄ 0.2% (〃) Glucose 1.0% (〃) Fructose 1.0% (〃) Cabicidin 100ppm (〃) Agar 1.5% (〃) ) PH6.2 Cultivated under anaerobic conditions at 30 ° C. for 2 to 3 days, streaking the emerged colonies on the agar plate medium, and culturing in the same manner,
Pure separation was performed.

After pure separation, apply again to the agar plate medium,
Microscopic observation was performed to confirm purification.

The morphological properties of the fungal part, properties on each medium, and physiological properties are as described above, and the present inventor named them Zymomonas mobilis subspecies mobilis B53.

Zymomonas mobilis subspecies mobilis
B69 was similarly isolated from sugar cane juice from Brazil.

As a result of the above test, it can be seen that the two strains of the microorganism of the present invention are strains that are purely isolated from nature.

Reference Example 1 Using the strain obtained in Example, ethanol was produced from molasses. Incidentally, the recovery of ethanol from the culture broth is carried out by using known means such as distillation, extraction, membrane separation and the like.

After inoculating the medium having the composition shown in Table 1 with the strain and statically culturing at 30 ° C. for 24 hours, 10% (v / v)% of this culture solution was inoculated into the medium having the composition shown in Table 2. Incubate at 30 ℃ for 24 hours. This culture solution is called a pre-culture solution.

Add this pre-culture liquid to a molasses dilution with a sugar concentration of 200 g / liter.
Inoculate 0 (v / v)% and incubate at 30 ℃ or 37 ℃.

The molasses fermentation characteristics at a culture temperature of 30 ° C are shown in Fig. 1.
Fig. 2 shows the molasses fermentation characteristics at ℃ (sugar concentration is 20
(W / v)%), and the ethanol concentration in the mash is shown in Table 3.

It can be seen that the strain according to the present invention has a higher ethanol production rate from molasses than known Zymomonas bacteria and is less susceptible to fermentation inhibition even under high temperature conditions.

Table 1 Medium composition yeast extract 1.0% (w / v) Glucose 2.0% (〃) pH unadjusted Table 2 Medium composition yeast extract 1.0% (w / v) Glucose 5.0% (〃) pH unadjusted Reference Example 2 Zymomonas mobilis B53 strain and B69 strain according to the present invention
Yeast extract (Oxoid) 1.0 (w
/ v)%, monopotassium phosphate 0.2 (w / v)%, glucose 2.
RM Slope Medium (pH: 0 (w / v)%, Agar: 2.0 (w / v)%)
One platinum loop was inoculated into 6.5) and cultured at 30 ° C for 24 hours. From this slant, one platinum loop was inoculated into a liquid medium (RM liquid medium) having the same composition, and static culture was carried out at 30 ° C. for 24 hours to prepare a pre-preculture medium. This pre-pre-preculture medium was again added to 1 part (volume) with respect to 100 parts (volume) of RM liquid medium, and static culture was carried out at 30 ° C. for 18 hours to obtain a pre-culture medium.

Add 1 part (volume) of this preculture liquid to 100 parts (volume) of RM liquid medium having a glucose concentration of 5.0 (w / v)% containing 0 to 3 (w / v)% KCl, and add at 30 ° C. The cells were statically cultivated for 3 days. After culturing, pass the culture solution through a filter with 0.45μ pore size,
The ethanol concentration was measured using a high performance liquid chromatograph or a gas chromatograph.

A similar experiment was performed on NR, a known bacterium of Zymomonas mobilis.
The RL B-14023 strain and the ATCC 29191 strain were also tested.

The results of 5 experiments are summarized in the following table.

Reference Example 3 In this example, strains B53 and B69 of the present invention and known strain NRRL B-140 are used.
The difference between the 23 strains and the ATCC 29191 strain was examined by focusing on the plasmids they carry.

The plasmid DNA of each strain was prepared by the alkaline extraction method of Birnboim and Doly (Nucleic acids Res. 7 , 1513 (1979)), purified with phenol / chloroform extraction and ethanol precipitation, and then purified with TE buffer (N- It was dissolved in tris (hydroxymethyl) aminomethane 10 mM + ethylenediaminetetraacetic acid 1 mM, pH 8.0).

Regarding each plasmid and its HindIII (Takara Shuzo) degradation product, λDNA-HindIII / φ × 174RF-HaeIII
The molecular weight marker (Pharmacia Co., Ltd.) was subjected to agarose gel electrophoresis according to a conventional method. It ’s
Electrophoresis was performed on the plasmid itself at an agarose gel concentration of 0.7% and at 1.0% for the HindIII digest of the plasmid and the above markers.

The B53 strain had a low molecular weight plasmid of about 5 kb and a plasmid of several tens of kb or more, whereas B69 had only a plasmid of several tens of kb or more. On the other hand, change the plasmid to Hi
The electrophoretic patterns of the digested fragments digested with ndIII were clearly different between the B53 and B69 strains, and these were the type cultures of Zymomonas mobilis NRRL.
It was also clearly different from the plasmids contained in B-14023 and ATCC 29191.

[Brief description of drawings]

The drawing is a graph showing the molasses fermentation characteristics of four strains. Fig. 1 shows the fermentation temperature of 30 ℃, and Fig. 2 shows the same temperature of 37 ℃.
It shows the case of.

Claims (3)

[Claims] 1. A liquid medium comprising glucose 5.0 (w / v)%, monopotassium phosphate 0.2 (w / v)%, yeast extract 1.0 (w / v)% and potassium chloride 2.0 (w / v)%. Zymomonas mobili that produces at least 1.75 (w / v culture solution)% ethanol when statically cultivated at a level of 10 ⁵ cells / ml at 30 ° C for 3 days at pH 6.5).
s). 2. At least 1.90 (w / w) when statically cultivated in the above liquid medium at a level of 10 ⁵ cells / ml at 30 ° C. for 3 days.
v Culture fluid) Zymomonas mobilis subspecies mobilis B53 (Zymomonas
mobilis subsp. mobilis B53: Microorganisms Research Institute No. 8922), Zymomonas mobilis according to claim 1. 3. At least 1.75 (w / w) when statically cultivated in the liquid medium at a level of 10 ⁵ cells / ml at 30 ° C. for 3 days.
v Culture fluid)% ethanol producing Zymomonas mobilis subspecies mobilis B69 (Zymomonas
Zymomonas mobilis according to claim 1, which is mobilis subsp. mobilis B69: Microorganisms Research Institute No. 8923).