JP2009060836A - Heat-resistant bacterium for producing ethanol and ethanol production method using heat-resistant bacterium for producing ethanol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new ethanol production bacterium strain for efficiently fermenting and producing ethanol under high-temperature conditions of 35°C or more, preferably 37-39°C and to provide an ethanol production method using the same. <P>SOLUTION: The wild isolated strains of Zymomonas mobilis, namely TISTR405 strain having accession number represented by NITE AP-440, and TISTR550 strain having accession number represented by NITE AP-441, are selected through a screening step for cultivation under high temperature condition and for examination of ethanol productivity. The ethanol production method includes using these strains. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel microorganism capable of producing ethanol under high temperature conditions and an ethanol production method using the same. More specifically, the present invention relates to a bacterial strain belonging to the Zymomonas mobilis species having ethanol-producing ability under a temperature condition of 37-39 ° C. and an ethanol production method using the same.

  Regardless of food or industrial use, ethanol is a substance used for various purposes. Chemically synthesized ethanol is used for industrial use, and brewed ethanol using microorganisms is used for food use. In recent years, due to the development of biotechnology and increased awareness of biomass utilization, attempts to use fermentation production technology not only for food but also for industrial use, particularly for ethanol for fuel, have begun to spread. Fermentation production technology using microorganisms for ethanol production is a technology that has been used for a long time, and many improvements aimed at improving the productivity of microorganisms that play a major role are also disclosed.

The main microorganism used for ethanol production is yeast (Saccharomyces cerevisiae), but it is known that only other bacteria belonging to the genus Zymomonas mobilis such as Zymomonas mobilis have ethanol fermentation ability. (Non-Patent Document 1). Zymomonas is a gram-negative anaerobic bacterium used for the production of Tequila and Pulque. In ethanol fermentation, its fermentation rate and ethanol production capacity per cell are larger than those of yeast, and it is produced during fermentation. It has advantageous features such as less biomass than yeast, but there are parts that are inferior to yeast, such as ethanol tolerance, temperature tolerance, and limited available sugar, and industrial use has not progressed. The current situation was. In recent years, fermentation production techniques using zymomonas bacteria include fermentation production techniques for useful substances such as levan (Patent Documents 1-5), various techniques including gene transfer for improving ethanol fermentation production (patents) Literature 6-9) and strains having useful characteristics such as salt tolerance, conjugation ability, and utilization improvement (Patent Literatures 10-12) have been disclosed so far, but are industrially important in fermentation production. The development of heat resistance, one of the improvements, has not progressed so far. Patent Document 8 describes that zymomonas and lactic acid bacteria are co-cultured and fermented at a high temperature of 37 ° C., which is high-temperature fermentation for keeping ethanol production low, and ethanol that is highly efficient under high-temperature conditions. It was not intended for production. Under these circumstances, the development of a heat-resistant microorganism that can be applied in ethanol fermentation production technology, for which demand is expected to increase rapidly, has been awaited.
Method for producing gluconic acid and sorbitol New strain of Zymomonas mobilis and method for producing levan using the strain Method for producing saturated delta-lactone using microorganism Cosmetic composition containing levan having cell proliferation effect and skin moisturizing and stimulating effect JP, 2004-339078, A preparation for external use Method for fermentation of alcohol Heterogeneous gene high expression vector Patent application title: Method for producing fermented milk containing appropriate amount of carbonic acid and low concentration of ethanol Patent application title: chromosomally integrated mannose fermenting zymomonas bacterium Bacteria suitable for ethanol production from molasses Patent application title: New Zymomonas mobilis New bacterium suitable for ethanol production from maltose Rogers P.M. L. et al. 1980. Process. Biochem. 15 (6): 7-11. Adachi O. et al. 1978. Agric. Biol. Chem. 42: 2045-56. Sambrook J. et al. & Russell DW. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor.

  In view of the above-mentioned present situation, the present invention is a novel zymomonas bacterium strain capable of efficiently producing ethanol under a temperature condition of 35 ° C. or higher, preferably 37-39 ° C., and ethanol production using the same. It is an object to provide a method.

  In order to solve the above problems, the present inventors have focused on fermenting microorganisms that always live in tropical regions under high temperature conditions, and isolated a Zymomonas mobilis (hereinafter abbreviated as Zm bacteria) strain that can solve this problem. Tried how to do. In other words, for several strains of Zm bacteria isolated and established from the tropical region, the growth ability and ethanol production ability at 30 ° C to 40 ° C were compared with the established high ethanol production strain of Zm bacteria. Among them, it was found that specific strains had high growth ability and ethanol fermentation production ability even under severe temperature conditions for Zm bacteria of 35 ° C. or higher, preferably 37-39 ° C., and the present invention was completed.

  That is, the first aspect of the present invention provides a thermostable ethanol-producing bacterium belonging to the Zymomonas mobilis species, which has an ethanol production ability under a temperature condition of 35 ° C. or higher.

  The second aspect of the present invention provides the thermostable ethanol-producing bacterium according to the first aspect, wherein the deposit number is represented by NITE AP-410 (TISTR405 strain).

  The third aspect of the present invention provides the thermostable ethanol-producing bacterium according to the first aspect, wherein the deposit number is represented by NITE AP-411 (TISTR550 strain).

  According to a fourth aspect of the present invention, there is provided an ethanol production method characterized by using the thermostable ethanol-producing bacterium according to any one of the first to third aspects.

  The fifth aspect of the present invention is characterized in that 1-5% (w / v) ethanol is produced under a temperature condition of 37-39 ° C. using a medium containing 3-20% glucose. The ethanol production method according to the fourth aspect is provided.

  By using the thermostable ethanol-producing bacterium provided by the present invention, it is possible to efficiently produce ethanol regardless of food or industrial use. In particular, the strain provided by the present invention preferably has the ability to efficiently produce ethanol by fermentation even under high temperature conditions of 37-39 ° C., reducing the cooling cost of the fermentation apparatus, and contamination of other types of microorganisms. It is expected that the cost reduction and efficiency improvement of ethanol fermentation production can be achieved, such as reducing the production rate and increasing the fermentation rate. Ethanol-producing bacteria have higher ethanol production efficiency than yeast normally used for ethanol production, and also have properties suitable for industrial use such as the amount of biomass produced during fermentation is smaller than yeast. Furthermore, the thermostable ethanol-producing bacterial strain provided by the present invention can efficiently produce ethanol even under high temperature conditions of 35 ° C. or higher, and its utility value is considered high.

  The best mode for carrying out the present invention will be described below. The first aspect of the present invention provides a thermostable ethanol-producing bacterium of the Zymomonas mobilis species characterized by having an ethanol fermentation ability under a temperature condition of 35 ° C. or higher. The present invention aims at improving productivity in ethanol production using Zm bacteria, and screens natural isolates of Zm bacteria with a focus on heat resistance. The present invention provides a Zm strain isolated from Zm bacteria at an optimum temperature and having ethanol-producing ability even at 35 ° C. or higher, more preferably 37-39 ° C. The ethanol-producing ability here is preferably 1 to 5% (w / v) 1-5% (w / v) in culture using a medium containing glucose (or other equivalent assimilable sugars). The ability to produce w / v) ethanol. Culture conditions other than the temperature used for ethanol production may be appropriately selected from the conditions used for ethanol production using Zm bacteria so far, and the present invention is not limited thereto. For example, ethanol fermentation A standard medium for microorganisms, such as yeast extract (Yeast extract), peptone, YPD medium containing (D-) glucose, and the like are suitable. The thermostable Zm bacterium provided by the present invention is obtained by a screening process in which a field isolate is allowed to produce ethanol under temperature conditions from 30 ° C. to 40 ° C. As a specific example, the deposit number is A strain represented by NITE AP-410 (405 strain) or a strain represented by NITE AP-411 (550 strain) is preferable, and these strains are white in shape, swelled, center protruding, colony The surroundings of the mouse showed a trait that was smooth and morphologically consistent with Zm bacteria, and as shown in the examples below, at the gene (adhA) level, Z. It shows 98% homology and 100% amino acid sequence homology with the ZM4 strain which is a high ethanol production strain of mobilis sp. It has been identified as a strain belonging to mobilis species.

  The Zm strain provided by the present invention is a strain that has been isolated from the natural world as a strain having ethanol fermentation ability under high temperature conditions. For example, the present strain relating to ethanol fermentation and temperature tolerance is used by genetic engineering techniques. Variety improvement, such as modifying genes of other species, or introducing genes from other species, such as genes for cellulase or pectinase to assimilate biomass raw materials, and adding more useful traits Is also effective. As a technique used for gene modification or introduction, a technique usually used in the field of genetic engineering using microorganisms may be applied as appropriate, and the present invention is not limited thereto. Techniques such as modification and introduction of a new gene (s) using a plasmid vector are conceivable.

  The present invention also provides a method for producing ethanol using the thermostable ethanol-producing bacterium described in any one of the first to third aspects. Since the basic principle of ethanol fermentation, anaerobic glycolysis, does not change even when the Zm strain provided by the present invention is used, an appropriate amount of ethanol is required for fermentative production of ethanol using the strain of the present invention. It is possible to perform fermentation by adding the ethanol-producing yeast of the above-mentioned aspect, with sugar (hydrocarbon) as the main component. The apparatus used for fermentation, equipment, the composition of the raw material liquid and the like may be appropriately used those used in the fermentation industry field, and do not limit the present invention. An ethanol production method using a contained culture solution, preferably a YPD culture solution, as a raw material solution is suitable. Moreover, as shown in the following Example, about 405 strains among the thermostable ethanol-producing bacterial strains provided by the present invention contain about 1-5% sugar content, which is said to maximize the fermentation efficiency in ethanol fermentation production. It is preferably used in a culture system (1% ethanol can be produced in a medium containing 3% glucose), while strain 550 is used in a culture system having a high sugar concentration exceeding 10% such as molasses (4 in a medium containing 16% glucose). % Ethanol can be produced), and it is preferably used according to the properties, raw materials, purpose, etc. of the strain.

  In ethanol fermentation production using heat-resistant ethanol-producing bacteria provided by the present invention, as a main component of sugar contained in the raw material liquid, as a raw material for alcoholic beverages in addition to pure sugars such as glucose, sucrose and fructose and mixtures thereof Various raw materials generally referred to as biomass, such as rice, wheat, straw, sugar cane, sugar maple, agave, sugar beet, can be used. Among these, those containing assimilable monosaccharides can be used as they are, and those containing mainly polysaccharides such as starch and cellulose can be used with enzymes that decompose these polysaccharides. It is preferable to use it, or to make a polysaccharide having hydrolysis ability hydrolyze the polysaccharide to decompose it into an assimilated monosaccharide. `` Bioethanol '' made from biomass raw materials is expected to increase in future demand as a fuel to replace petroleum resources, and by using the heat-resistant ethanol-producing bacteria provided by the present invention and ethanol produced by these bacteria, It is expected to contribute to bioethanol related industries. Examples of the present invention are shown below, but the present invention is not limited to the examples.

  (Zymomonas strain and culture) As experimental strains of Zymomonas mobilis, TISTR405, TISTR548, TISTR550, and TISTR551 strains (hereinafter, the numbers are used to represent the strain names) were used. Moreover, ZM4 strain | stump | stock (deposit number: NRRL B-14023) which is a ethanol high production strain of Zm bacteria was used as a control regarding the ethanol productivity of these strains. For the culture of Zm bacteria, 100 ml of YPD medium containing 0.3% yeast extract, 0.5% peptone, and 3% glucose was used, and shaking culture was performed at a speed of 100 rpm (Round per minute). The ethanol concentration in the culture solution was measured by the method described in Non-Patent Document 2, that is, a method using purified alcohol dehydrogenase derived from acetic acid bacteria. The growth of Zm bacteria was calculated by measuring the OD600 (absorbance at 600 nm) of the culture solution.

  (Species identification of strain) TISTR405 strain, 548 strain, 550 strain, 551 strain form colonies on YPD agar medium, and when looking at the shape, the characteristics are white, bulging, and the central portion is protruding, Was smooth. This was consistent with the characteristics of the Zymomonas mobilis species. For further identification, a fragment of the alcohol dehydrogenase gene (adhA) was cloned from 405 genomic DNA of these strains, and the nucleotide sequence was compared with a database of known species. Preparation of genomic DNA from Zm bacteria was performed by the method described in Non-Patent Document 3, Primers (SEQ ID NOs: 1 and 2) were designed with reference to the adhA base sequence of ZM4 strain, which is a high ethanol production strain of mobilis species, and a 1630 bp gene fragment containing about 500 bp upstream of the adhA coding region was obtained by PCR. . The PCR product was introduced into a pUC119 plasmid vector, and the nucleotide sequence was determined using M13 universal primers (SEQ ID NOs: 3 and 4). This represents the sequence revealed in SEQ ID NO: 5. When this sequence was used to perform a homology search using the NCBI database, Z. It was 98% homologous at the nucleotide sequence level with the adhA gene of mobilis ZM4 strain, and 100% completely matched when translated into amino acids. From these results, TISTR405 strain, 548 strain, 550 strain, and 551 strain provided by the present invention are Z. It was identified as a microorganism belonging to the species mobilis.

  (Examination of growth and ethanol production ability-3% glucose) In order to clarify the heat resistance of each strain of Zm bacteria and ethanol production ability under high temperature conditions, each strain was cultured at 30 to 40 ° C and its growth And the ethanol concentration in the culture broth was measured. Since all the strains did not grow at 40 ° C, the subsequent examination was conducted at a temperature of 39 ° C or lower. FIG. 1 shows the results of this study. FIG. 1A shows the growth of each strain at 30 ° C., the vertical axis represents the value of OD600, and the horizontal axis represents the time from the start of culture (hour). FIG. 1B shows ethanol production ability at 30 ° C., the vertical axis represents ethanol concentration (w / v), and the horizontal axis represents time from the start of culture (hour). In the graph,-○-indicates 405,-△-indicates 550,-□-indicates 551,-▲-indicates 548,-●-indicates ZM4 (symbol is A). Common to -D). As can be seen in FIG. 1A, 548 strains show a similar growth curve except that growth start is slightly slow, and 550 and 551 strains tend to grow slightly slower than other strains after 24 hours. Indicated. On the other hand, in terms of ethanol production ability (FIG. 1B), 550 strains showed the highest ethanol concentration of about 1.0% in the culture solution, and then 405,551 and Zm4 strains had an ethanol concentration of about 0.8%. showed that. It was judged that 548 strains were not suitable for ethanol production because the ethanol concentration in the culture broth hardly increased. On the other hand, although 550 strains did not grow so fast, ethanol production ability was the highest among all strains including ZM4 strain, which is a high ethanol production strain, indicating that it is suitable for ethanol production. .

  The examination result in 39 degreeC which is high temperature conditions is shown to FIG. FIG. 1C shows the growth of each strain at 39 ° C. (axis is common with A), and FIG. 1D shows the ethanol production ability at 39 ° C. (axis is common with B). As shown in FIG. 1C, at 39 ° C., the growth of the Zm4 strain, which is a high ethanol production strain, decreased to 0.5 or less as the OD600 value, and the 551 strain showed almost no growth. On the other hand, 405 strain showed good growth similar to 30 ° C. even at this temperature, and 548 and 550 strains also showed higher growth ability than the control ethanol high-producing strain. In comparison of ethanol production ability at 39 ° C., ZM4 strain showed only about 40% ethanol production ability of about 0.4% and 30 ° C., whereas 405 and 550 strains showed about 1% and 30%. Ethanol production ability similar to that at ℃, and 405 strain shows ethanol production ability better than 30% at 0.8% at 24 hours after the start of culture and 1% at 36 hours. It was shown to be possible. 550 strains, like 30 ° C, had a lower growth at 39 ° C than 405 strains, but the ethanol concentration in the culture broth was high, and it was an excellent strain from the viewpoint of ethanol production ability per cell. Indicated. The 551 strain showed an ethanol productivity of 0.2% after 60 hours, and the 548 strain showed almost no increase in ethanol concentration. From these results, it was revealed that not all Zm strains are suitable for the production of thermostable ethanol, and specific strains, ie, 405 and 550 strains are suitable for the production of thermostable ethanol.

  (Investigation of growth and ethanol production ability—16% glucose) In order to examine the growth ability and ethanol production ability of the strain of the present invention in a culture solution containing higher concentrations of glucose, a YPD medium containing 16% glucose (glucose concentration) Except for the above, the growth and ethanol production ability of 405 strain, 550 strain and ZM4 strain, which is a high ethanol production strain of the control, at 30 ° C. and 39 ° C. were examined. The growth ability was a value of OD600, and the ethanol production ability was calculated by measuring the ethanol concentration in the culture solution. The result of the examination is shown in FIG. FIG. 2A represents the growth of each strain at 30 ° C., the vertical axis represents the value of OD600, and the horizontal axis represents the time from the start of culture (hour). In the graph, -O- indicates 405 strains, -Δ- indicates 550 strains, and-●-indicates ZM4 strains (symbols are common to AD). In the culture with a glucose concentration of 16%, the OD600 value of the 405 strain was smaller than those of the other two strains. FIG. 2B shows ethanol production ability at 30 ° C., and about 3% ethanol was produced after 36 hours in the ZM4 strain. On the other hand, 4% ethanol was produced in 550 strains after 36 hours, and the ethanol production ability was superior to that in the ZM4 strain under these conditions.

  The growth ability and ethanol production ability at 39 ° C. are shown in FIGS. 2C and D, respectively. Similar to 30 ° C., regarding the growth (C), no difference was observed between the ZM4 strain and the 550 strain except that the OD600 value of the 405 strain was 1 or less. On the other hand, in the ethanol production capacity (D), the control ethanol high production strain, 405 strain, showed only 2% or less ethanol production ability, whereas 550 strain had an ethanol production capacity of nearly 4%, which was close to 30 ° C. In addition to the heat-resistant ethanol production ability, 550 strain was shown to have a high ethanol production ability even in a culture solution containing a high concentration of sugar.

  The ethanol-producing bacteria provided by the present invention can be used in the fermentation industry that uses microorganisms for the production of various useful substances. In particular, the ethanol-producing bacterium of the present invention has good ethanol-producing ability under severe high temperature conditions for zymomonas bacteria of 37 ° C. or higher, and has advantages such as cost reduction in ethanol production including bioethanol. Expected to bring. Further, from the examination results of Examples 1 and 2, when using the strain of heat-resistant ethanol-producing bacteria provided by the present invention, a culture solution containing about 3% of sugar, which is said to have the highest ethanol production efficiency, is used. 405 strains are suitable for conventional culture, and 550 strains are suitable for culture using a culture solution containing a high concentration of sugar such as molasses, such as molasses, and it is preferable to use them according to the purpose and application. It is done.

The growth (A, C) and ethanol production ability (B, D) of the thermostable ethanol-producing bacteria provided by the present invention at 30 ° C. and 39 ° C. in a system using a culture solution containing 3% glucose was determined as follows. -Shown in comparison with Mobilis high ethanol production strain. In a system using a culture solution containing 16% glucose, the growth (A, C) and ethanol production ability (B, D) of the thermostable ethanol-producing bacteria provided by the present invention at 30 ° C. and 39 ° C. were expressed as Zymomonas. -Shown in comparison with Mobilis high ethanol production strain.

Claims (5)

  A thermostable ethanol-producing bacterium belonging to the species Zymomonas mobilis, characterized by having ethanol-producing ability under a temperature condition of 35 ° C or higher.   The thermostable ethanol-producing bacterium according to claim 1, wherein the deposit number is represented by NITE AP-410 (TISTR405 strain).   The thermostable ethanol-producing bacterium according to claim 1, wherein the deposit number is represented by NITE AP-411 (TISTR550 strain).   A method for producing ethanol, comprising using the heat-resistant ethanol-producing bacterium according to any one of claims 1 to 3.   The ethanol production method according to claim 4, wherein 1% (w / v) or more ethanol is produced under a temperature condition of 37-39 ° C. using a medium containing 3% glucose.

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