JP5769187B2 - Microorganism having resolvability of cellulose raw material and method for decomposing cellulose raw material using the same - Google Patents

Description

  The present invention relates to a microorganism strain capable of decomposing a cellulose raw material (hereinafter also referred to as “cellulosic biomass”) and producing ethanol under a high temperature condition of 38 to 45 ° C., and a method for decomposing a cellulose raw material using the same.

  As a substitute for petroleum, bioethanol, which is produced by fermentation of microorganisms from various raw materials, is expected to contribute to global warming countermeasures as a so-called carbon neutral fuel, unlike fossil fuels. However, at present, in the ethanol production from cellulosic biomass that does not compete with food in particular, there are significant problems in terms of cost, and it has not reached industrialization.

  For the use of cellulosic biomass and bioethanol production from cellulosic biomass, the main component cellulose is a linear polymer formed by glycosidic bonds of glucose. Has been developed to produce glucose and ethanol fermentation using the obtained glucose as a sugar source. Cellulose decomposition methods mainly include chemical treatment methods, that is, a method of cleaving glycosidic bonds under conditions of high temperature, high pressure, and strong acid (Patent Document 1), and biological treatment methods, that is, cellulose decomposition such as cellulase. A method using a catalytic enzyme (Patent Document 2) is known.

  Since chemical treatment of cellulose is a treatment method with a high environmental load, development of a method for biologically degrading cellulose having a relatively low environmental load has been attempted. Cellulases derived from various organisms are typical examples, and cellulases isolated from bacteria (Patent Document 3), filamentous fungi (Patent Document 4), protozoa (Patent Document 5) and the like are disclosed.

  Furthermore, focusing on the microorganisms having the ability to decompose cellulose, a method of degrading cellulose into living microorganisms by so-called fermentation is also expected from the point that it can be reused, but it has not reached the industrial practical level. Is the current situation. As a method of degrading cellulose into microorganisms, there is a method of expressing by incorporating a cellulolytic enzyme derived from another species into a microorganism usually used for fermentation by gene recombination (Patent Document 6). It has not been realized from the viewpoint of release into the environment.

  On the other hand, the use of microorganism strains having cellulose resolving ability / cellulolytic enzyme-producing ability includes Bacillus sp. KSM-N257 strain (FERM P-17473; Patent Document 7), Acremonium cellulolyticus (Acremonium). cellulolyticus) C1 strain (FERM P-18508; Patent Document 8), Cellulomonas genus microorganism K32A strain (Patent Document 9), Bacillus genus microorganism KSM-330 (Microtechnological Bacteria No. 11223; Patent Document 10) and the like are disclosed. ing. Although each of them has a predetermined usefulness, it has not been possible to industrially decompose cellulose by microbial fermentation to produce glucose, or to produce ethanol from cellulose itself in a microbial fermentation process.

  In addition, in the fermentative production of useful substances using microorganisms, there is a problem that the heat of fermentation generated in the process of microorganisms metabolizing raw materials significantly reduces the activity of the fermentation microorganisms themselves. At present, in most cases, a cooling device is installed in the fermenter to solve this problem by keeping the temperature in the fermenter within a certain range, but from the viewpoint of cost reduction and energy saving, some systems A microorganism strain having a trait that maintains physiological activity even under a temperature condition higher than that normally applied for each bacterial species / strain (in the present invention, this trait is defined as “heat-resistant”) is used. Attempts have been made. However, such an approach has not been made in the technology relating to the fermentation decomposition of cellulosic biomass using microorganisms.

Patent No. 4330839 Method for producing glucose and / or water-soluble cellooligosaccharide Patent No. 3075609 Method for producing cellooligosaccharide Patent No. 4392778 Actinomycetes producing novel cellulases, cellulases produced by the actinomycetes, and methods for producing the cellulases Patent No. 3962805 Protein having cellulase activity and method for producing the same Patent No. 4224601 Cellulase gene derived from termite symbiotic protozoa Patent No. 4017824 Endoglucanase enzyme and cellulase preparation comprising the same Patent No. 4382994 Novel alkaline cellulase Patent No. 4025848 Method for decomposing cellulose raw material Patent No. 3710997 Novel microorganism, microorganism-producing enzyme, and plant fiber decomposition method using the enzyme Patent No. 2929023 Cellulase, microorganism producing the same, and method for producing cellulase

  In view of the above-mentioned present situation, an object of the present invention is to provide a microorganism strain capable of degrading a cellulose raw material by fermentation and capable of withstanding a temperature increase due to fermentation, and a method for decomposing the cellulose raw material using the same.

  In order to solve the above-mentioned problems, the present inventors pay attention to a microbial community in bovine rumen as a microflora having a cellulose-decomposing ability, and (1) from the microorganisms present here, only cellulose is used as a sugar source. (2) Microbial strains that can be cultured were searched through two screening processes, culturing in a high temperature range around 40 ° C. As a result, they found that a specific strain of Bacillus licheniformis, a kind of facultative aerobic Gram-positive gonococci, has a trait of cellulose resolution and high physiological activity at around 40 ° C., thereby completing the present invention. It was.

That first aspect of the present invention, Bacillus licheniformis (Bacillus characterized by having the following properties
licheniformis) strain having the accession number NITE P-891 and the Bacillus licheniformis R15 strain having the accession number NITE P-892 .
(1) Can survive and grow under the temperature of 38-45 ° C (2) Can produce cellulose by decomposing cellulose (3) Can produce ethanol from cellulose raw material as sugar source

The second aspect of the present invention provides a method for decomposing a cellulose raw material using the Bacillus licheniformis strain described in the first aspect .

A third aspect of the present invention provides a method for producing ethanol using the Bacillus licheniformis strain described in the first aspect .

  Regarding Bacillus bacteria, enzymes having heat resistance have been isolated in some cases (Japanese Patent Laid-Open No. 2006-149374, Japanese Patent Laid-Open No. 2001-057852, Special Table 2002-529610), and are not Bacillus bacteria. A thermostable cellulase and a microorganism strain producing the same (Patent No. 3512981, Japanese Patent Publication No. 7-2113) have also been presented. The term “thermostable” in these inventions refers to the fact that Bacillus bacteria enter a dormant state. It is derived from the observation result that the spore can withstand a high temperature of 60 ° C. or higher, and is heat resistant in the sense that the enzyme itself does not lose its activity in a temperature range of 60 ° C. or higher. In the present invention, the invention itself is based on a technical idea different from “heat resistance” in the sense that the microorganism itself does not lose its physiological activity under high temperature conditions.Furthermore, regarding the Bacillus licheniformis species, as shown in the examples below, the above-mentioned “heat-resistant” is not observed in the standard strain because growth using a cellulose raw material as a sugar source, growth at 38 ° C. or higher, and ethanol production ability are not recognized. Neither the property, cellulose resolving power nor ethanol-producing ability is generally recognized by Bacillus bacteria, indicating that the strain of the present invention has outstanding properties among Bacillus licheniformis species.

  By utilizing the present invention, it becomes possible to efficiently perform decomposition treatment of cellulosic biomass and ethanol production from cellulosic biomass under a high temperature condition around 40 ° C. Since a large part of the cost in the fermentation production of useful substances is attributed to the cooling cost of fermentation heat, the present invention enables bioethanol production from a cellulose raw material with particularly reduced cooling cost.

The screening result of heat-resistant cellulose decomposing bacteria is shown. The cellulose resolution of heat-resistant cellulose-decomposing bacteria is shown for each strain. The cellulose resolution under stationary and stirring conditions is shown for each strain. The cellulose resolution in the existing microbial strain with a cellulose resolving power, the Bacillus licheniformis standard strain, and the strain of the present invention is shown in comparison. The growth at 42 ° C. and cellulose degradation over time in the Bacillus licheniformis standard strain and the strain of the present invention are shown in comparison. The growth at 42 ° C. and the time course of ethanol production in the Bacillus licheniformis standard strain and the strain of the present invention are shown in comparison.

  The form for implementing this invention is shown below. The present invention is capable of surviving and growing under a high temperature condition of 38-45 ° C., more preferably 40-45 ° C., and capable of decomposing cellulose raw materials (degrading cellulose to produce reducing sugar). A Bacillus licheniformis strain having a trait is provided. As a preferred embodiment, specific strains include R1, R2, R3, R4, R5, R6, R7, R8, R9, R10. , R11, R12, R13, R14, R15, R16, a more preferred embodiment is a Bacillus licheniformis R8 strain deposited with a deposit number of NITE P-891, or a deposit number is Bacillus licheniformis R15 bacteria deposited with NITE P-892 Offer stocks.

  Bacillus licheniformis is a kind of Gram-positive bacilli that is commonly observed in soil and bird feathers and is also known as a causative agent of food spoilage. The optimum temperature for growth under culture conditions is around 30 ° C. (Bacillus licheniformis), and attention has not been paid to the relationship with cellulose degradation and the relationship with bioethanol production. As shown in the following examples, in comparison with Trichoderma reesei, a kind of filamentous fungus having cellulose resolving power, Trichoderma exhibits higher cellulose resolving power than R8 and R15 strains at a commonly used temperature of 30 ° C. However, the present inventors have shown that this is reversed at 40 ° C., and that the cellulose resolving power of Trichoderma is almost 0 at 41-42 ° C., whereas the R8 and R15 strains show vigorous cellulose resolving power at this temperature. Furthermore, as shown in the following examples, regarding the cellulose resolving power of the strain provided by the present invention, the stationary condition shows a cellulose resolving power that is about 4-12 times higher than the stirring condition that is usually used. In particular, cells in specific strains of Bacillus licheniformis There is a possibility that the potential for over scan decomposition and heat resistance have been overlooked.

  In the present invention, R8 and R15 strains were isolated by screening microbial communities present in bovine rumen and culturing under a temperature condition around 40 ° C. using only cellulose as a sugar source. In a previous study on microorganisms and cellulose that are normally present in the world (Handa et al., Proceedings of the 20th Annual Meeting of the Japanese Society for Microbial Ecology 20 pp39), Butyrivibrio fibrisolvens, Prevoterra, ), Luminobacter amylophilus, Corynebacterium vitavita. rumen) and bacteria of unknown lineage have been reported, but there has been no report that links Bacillus licheniformis to rumen and cellulose degradation, and that the point of focus of the present invention was different from the points of focus so far. Show.

  The Bacillus licheniformis R8 strain provided by the present invention has the base sequence of SEQ ID NO: 1 in the base sequence of 16s rDNA, can survive at 45 ° C., and is cultured when carboxymethyl cellulose (CMC) is used as the sole sugar source. Can produce about 0.4 mg / ml reducing sugar in 48 hours, about 0.85 mg / ml reducing sugar in 72 hours, and about 0.0055% ethanol in 48 hours using cellulose raw material as a sugar source Is a strain having the characteristic that about 0.0065% ethanol can be produced in 72 hours.

  The R15 strain (deposit number: NITE P-892) in the embodiment of the present invention is also a strain isolated by the same screening as the R8 strain in the above embodiment, and the base sequence described in SEQ ID NO: 2 in the base sequence of 16s rDNA And can survive at 45 ° C. When cultured with CMC as the sole sugar source, it can produce approximately 0.3 mg / ml of reducing sugar in 48 hours and 0.7 mg / ml of reducing sugar in 72 hours. And about 0.0035% ethanol in 48 hours and about 0.005% ethanol in 72 hours using a cellulose raw material as a sugar source.

  The cellulose raw material that can be used by the strain provided by the present invention may be any fibrous raw material mainly composed of cellulose synthesized by plants, such as plant cell wall components other than cellulose, for example, Hemicellulose, lignin, etc. may be included. In addition, the shape of the plant may be any material that is mainly composed of cellulose, such as a physically crushed plant body, pulp grounds, processed vegetable residue, pagas, waste wood chips, rice straw, waste paper, etc. There may be.

The method for decomposing cellulose raw material using the strain provided by the present invention refers to a method for fermentatively decomposing cellulose raw material using the strain provided by the present invention, regardless of direct or indirect, and its form is not limited. . The cellulose raw material decomposition method using the present strain also includes a method for producing a saccharide (preferably a monosaccharide, more preferably a monosaccharide that is a reducing sugar) from the cellulose raw material. Among these, in particular, the strain provided by the present invention is suitable for a method for decomposing a cellulose raw material under stationary conditions without stirring the reaction solution.
The saccharide derived from the cellulose raw material obtained in the above embodiment can be used as a raw material for combining with other fermentation processes.

The strain provided by the present invention is also applicable to a method for producing ethanol, more preferably a method for producing ethanol from a cellulose raw material. The ethanol production method here includes a process from saccharification of raw materials to ethanol fermentation, and any process is an ethanol production method using the strain provided by the present invention. It should be included in the invention. At this time, available modes can be broadly divided into (A) a cellulose raw material to decompose to produce saccharides, and the obtained saccharides can be used as other types of ethanol-fermenting microorganisms, preferably yeasts such as Saccharomyces and Kliberomyces, and Zymomonas bacteria. As a substrate for ethanol production by zymobacter bacteria, two modes of (B) producing ethanol from a cellulose raw material using a suitable strain provided by the present invention can be used. In the embodiment of (A), there are a step of fermenting and decomposing a cellulose raw material using the strain of the present invention, and a step of performing ethanol fermentation using the saccharide obtained from the cellulose raw material as a raw material and using the other types of ethanol-fermenting microorganisms. Although contemplated, this may be in the same fermentor or in separate fermenters. On the other hand, in (B), it is possible to perform ethanol fermentation directly from a cellulose raw material using a suitable strain provided by the present invention in the same fermentor.
Examples of the present invention are shown below, but the present invention is not limited to the examples.

  (Screening of strains) Rumen fluid was collected from cattle (beef cattle, females) raised on Yamaguchi University-affiliated farm, 0.2% carboxymethylcellulose (CMC), 0.67% yeast nitrogen base w / o amino acids. 10 μl was spread on a CMC agar plate containing ammonium sulfate, 0.02% peptone and 1.7% agar and grown overnight at 38 ° C., 39 ° C., 40 ° C., 41 ° C., 42 ° C., 45 ° C. When the colonies generated on each plate were counted, the results shown in FIG. 1A were obtained. Among these colonies, colonies that grew at 42 ° C. were named R1 to R8, and colonies that grew at 45 ° C. were named R9 to R16, respectively, as strains having higher heat resistance. These colonies were spotted again on the above-mentioned CMC agar plate and cultured overnight at 45 ° C. FIG. 1C shows that all the strains grew well under this temperature condition. FIG. 1B shows the result of staining this plate with a staining solution prepared by dissolving 2 g of potassium iodine and 1 g of iodine in 300 ml of distilled water. Although the cellulose component is dyed dark by this dyeing, no dyeing occurs because the cellulose component is decomposed in the portion where the colonies are formed.

(Carboxymethylcellulose resolution of heat-resistant cellulose-degrading bacteria) 5 ml of a liquid medium containing 2% carboxymethylcellulose, 0.67% yeast nitrogen base w / o amino acid / ammonium sulfate, 0.02% peptone And inoculated to 42 ° C. for 72 hours. After 48 hours from the start of the culture, the medium after 72 hours was collected, and a medium supernatant was obtained with a centrifuge. The reducing sugars of these culture supernatants were measured by the dinitrosalicylic acid method (DNSA method; Miller 1959. Anal. Chem 31).
In FIG. 2, the CMC resolution of each strain obtained by screening is compared and shown. In the figure, R1, R2,... Represent each strain, and the vertical axis represents the amount of reducing sugar (mg / ml) in the culture solution, which is an indicator of CMC resolution. The white bar graph represents the results for 48 hours after the start of the culture, and the black bar graph represents the results for 72 hours. As shown in the graph, when focusing attention on the amount of reducing sugar after 72 hours, the R8 strain was the largest at 0.8 mg / ml, and then the R15 strain was about 0.75 mg / ml.

(Comparison under standing and stirring conditions) Each of the above strains was inoculated into 5 ml of a liquid medium containing 2% carboxymethylcellulose, 0.67% yeast nitrogen base w / o amino acid / ammonium sulfate, 0.02% peptone. The mixture was grown at 42 ° C. for 48 hours under the conditions of standing and stirring. The culture medium after 48 hours was collected, and a culture supernatant was obtained with a centrifuge. The reducing sugars in these medium supernatants were measured by the dinitrosalicylic acid method.
FIG. 3 shows a comparison of CMC resolution of each strain under static and stirring conditions. In the figure, R1, R2... Indicate each strain, the vertical axis indicates the amount of reducing sugar (mg / ml) in the culture solution, the light gray bar graph indicates the results under static conditions, and the dark gray bar graph indicates the stirring conditions. Each result is shown below. As the graph shows, CMC was decomposed better in the standing condition than in the stirring condition for all the strains, and in particular, the R7 strain (about 0.43 mg / ml), the R8 strain (about 0.4 mg / ml), the R15 strain The activity was high at 0.3 mg / ml.

  The results in FIGS. 2 and 3 were combined, and the R7 strain had a small amount of reducing sugar after 72 hours. Therefore, the strains R8 and R15 were used for verification as strains having high cellulose resolution. In addition, when genomic DNA was extracted from these strains and the base sequence of 16s rDNA was determined according to a conventional method, the R8 strain had the base sequence of SEQ ID NO: 1, and the R15 strain had the base sequence of SEQ ID NO: 2, respectively. It became clear. When these base sequences were used with BLAST and compared with existing ones, they almost coincided (99% or more) with bacteria of the Bacillus licheniformis species, so this strain was identified as a Bacillus licheniformis species.

(Comparison with other strains) Cellulose degradability exhibited by the R8 and R15 strains was compared with other strains of the same species and cellulose-degrading bacteria of other species. The Bacillus licheniformis NBRC12220 strain (a standard strain of the same species) was selected as a comparison target of the same species, and the Trichoderma reesei NBRC31329 strain of a filamentous fungus was selected as a comparison target of other species.
Each strain was inoculated into 5 ml of a liquid medium containing 2% carboxymethylcellulose, 0.67% yeast nitrogen base w / o amino acid / ammonium sulfate, 0.02% peptone, 30 ° C, 37 ° C, 39 ° C, 40 ° C, The cells were grown for 72 hours at 41 ° C and 42 ° C. The culture medium after 72 hours was collected, and a culture supernatant was obtained with a centrifuge. The amount of reducing sugar in these medium supernatants was measured by the dinitrosalicylic acid method.

  In FIG. 4, the comparison result with the strain of this invention and another strain is shown. The horizontal axis of the graph indicates each temperature condition (sta indicates the stationary condition), the vertical axis indicates the amount of reducing sugar (mg / ml) in the culture solution, the white bar graph indicates the R8 strain, the black bar graph indicates the R15 strain, and light gray These graphs represent the results of Trichoderma reesei NBRC 31329 strain, and the dark gray graphs represent the results of Bacillus licheniformis NBRC12200 strain. On the left side of the graph, at 30 ° C. and 37 ° C., Trichoderma showed the highest cellulose resolution, while the standard strain of Bacillus licheniformis showed no cellulose resolution at all. On the other hand, the cellulose resolving power of the two strains of the present invention and Trichoderma was almost the same at 39 ° C., and reversed at 40 ° C. or higher. In particular, at 41 ° C. and 42 ° C., Trichoderma hardly decomposes cellulose, whereas the strain of the present invention maintains vigorous cellulose resolving power, and shows high cellulose resolving power of the strain of the present invention under high temperature conditions. It was.

  (Change over time of growth and cellulose degradation of R8, R15 strains and ethanol production) Growth and cellulose degradation of the strain of the present invention, and changes over time in growth and ethanol production were compared with the Bacillus licheniformis standard strain (NBRC12200 strain). Compared. Each strain was inoculated into 30 ml of a liquid medium containing 2% carboxymethylcellulose, 0.67% yeast nitrogen base w / o amino acid / ammonium sulfate, 0.02% peptone, and allowed to grow for 72 hours at 42 ° C. . The culture medium after 0, 24, 48, and 72 hours was collected, and the culture supernatant was obtained with a centrifuge. The amount of reducing sugar contained in these medium supernatants was measured by the dinitrosalicylic acid method, and the amount of ethanol was measured by a method using purified alcohol dehydrogenase derived from acetic acid bacteria (Adachi et al. 1978. Agric. Biol. Chem. 42). .

  FIG. 5 shows changes over time in growth and cellulose degradation. The horizontal axis of the graph is the time from the start of culture, the left vertical axis is the OD600 value as a growth index (corresponding to the bar graph), and the right vertical axis is the amount of reducing sugar in the culture solution (corresponding to the mg / ml line graph). The white bar graph shows the growth of the R8 strain, the black bar graph shows the growth of the R15 strain, the light gray bar graph shows the growth of the standard strain. Degradation and broken lines -Δ- represent cellulose degradation of the standard strain, respectively. As the graph shows, at this temperature (42 ° C.), the standard strains show some growth by 24 hours but decrease in 48-72 hours, whereas the two strains of the present invention, particularly the R8 strain, are high. It showed proliferative ability. Although the growth ability of the R15 strain is slightly inferior to that of the R8 strain, after 72 hours, the amount of reducing sugar in the culture becomes almost the same as that of the R8 strain, and shows excellent properties in terms of cellulose resolution per cell. It was. In this system, the standard strain did not degrade the cellulose at all, confirming the peculiarity of the strain of the present invention.

FIG. 6 shows changes over time in growth and ethanol production. The horizontal axis of the graph represents the time from the start of culture, the left vertical axis represents the OD600 value as a growth index (corresponding to the bar graph), the right vertical axis represents the ethanol concentration in the culture solution (corresponding to the% line graph), white The bar graph shows the growth of the R8 strain, the black bar graph shows the growth of the R15 strain, the light gray bar graph shows the growth of the standard strain, the line-○-indicates the ethanol production of the R8 strain, the line-●-indicates the ethanol production of the R15 strain, the line -△-represents ethanol production of the standard strain, respectively. As shown in the graph, at this temperature, the R8 strain exhibits high ethanol production ability using only cellulose as a sugar source, about 0.002% in 24 hours, about 0.0055% in 48 hours, and about 0.00 in 72 hours. It was 0065%. On the other hand, the R15 strain also exhibited ethanol-producing ability although the start-up was slightly slow, about 0.003% in 48 hours and about 0.005% in 72 hours.

Claims (3)

Bacillus licheniformis strain characterized by having the following properties and deposited with Bacillus licheniformis R8 strain deposited under NITE P-891 or deposited under NITE P-892 Bacillus licheniformis R15 strain .
(1) Can survive and grow under the temperature of 38-45 ° C (2) Can produce cellulose by decomposing cellulose (3) Can produce ethanol from cellulose raw material as sugar source A method for decomposing a cellulose raw material using the Bacillus licheniformis strain according to claim 1 . A method for producing ethanol using the Bacillus licheniformis strain according to claim 1 .