阅读说明：本技术 产氢气菌及其用于生产氢气的方法 (Hydrogen producing bacterium and method for producing hydrogen by using same ) 是由 魏小波 魏洪炎 程丽楠 马井阳 臧灵霞 靳辉 于 2021-09-18 设计创作，主要内容包括：本发明提供一种产氢气芽孢杆菌(Bacillus sp.)JBW1-26a,其保藏编号为CGMCC NO:22807、产氢气梭菌(Clostridium sp.)JBW1-26b,其保藏编号为CGMCC NO:22808,以及一种生产氢气的方法,其包括使用产氢气芽孢杆菌(Bacillus sp.)JBW1-26a和/或产氢气梭菌(Clostridium sp.)JBW1-26b进行发酵来生产氢气。与现有技术相比,本发明的产氢气菌具有以下优势：对环境不敏感,有无光照、有无氧气、二氧化碳等气体均不影响产氢过程,可利用底物来源广泛,采用农作物的副产品或副产品的二次加工产品即可作为底物,产氢效率高,与厌氧发酵产氢细菌相近,产气过程中氢气含量高。(The invention provides hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a with a preservation number of CGMCC NO. 22807, hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b with a preservation number of CGMCC NO. 22808, and a method for producing hydrogen, which comprises fermenting with the hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a and/or the hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b to produce the hydrogen. Compared with the prior art, the hydrogen-producing bacterium has the following advantages: the method is insensitive to the environment, has no influence on the hydrogen production process due to the existence of light, oxygen, carbon dioxide and other gases, can utilize wide substrate sources, can use byproducts of crops or secondary processing products of the byproducts as the substrate, has high hydrogen production efficiency, is similar to anaerobic fermentation hydrogen production bacteria, and has high hydrogen content in the hydrogen production process.)

1. A hydrogen producing Bacillus (Bacillus sp.) has 16s rRNA sequence shown in SEQ ID NO. 1.

2. The hydrogen-producing Bacillus (Bacillus sp.) according to claim 1, which is numbered JBW1-26a and has a preservation number of CGMCC NO. 22807.

3. A Clostridium hydrogenogenum (Clostridium sp.) has a 16s rRNA sequence shown in SEQ ID NO. 2.

4. Clostridium hydrogenogenum (Clostridium sp.) according to claim 3, having the accession number JBW1-26b and the accession number CGMCC NO 22808.

5. A method for producing hydrogen comprising producing hydrogen by fermentation using a hydrogen-producing microorganism, wherein the hydrogen-producing microorganism is Bacillus hydrogengenes (Bacillus sp.) JBW1-26a of claim 1 or 2 and/or Clostridium hydrogengenes (Clostridium sp.) JBW1-26 of claim 3 or 4; preferably the hydrogen-producing microorganisms comprise hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a and hydrogen-producing Clostridium (Clostridium sp.) JBW1-26 b; further preferably, the ratio of hydrogen producing Bacillus (Bacillus sp.) JBW1-26a to hydrogen producing Clostridium (Clostridium sp.) JBW1-26b is 0.1-10: 1.

6. The method of claim 5, wherein the method comprises:

activating the hydrogen-producing microorganisms, and inoculating the activated hydrogen-producing microorganisms into a seed culture medium for culture to obtain a seed solution;

and inoculating the seed solution into a fermentation culture medium containing a substrate for fermentation culture to obtain hydrogen.

7. The method of claim 6, wherein the seed medium comprises: 5.0-12.0g/L of peptone, 1-6g/L of beef extract powder, 4-6g/L of sodium chloride and 7.0-7.5 of pH value.

8. The method according to claim 6, wherein the fermentation temperature is 25-45 ℃ and the fermentation time is 60-120h in the fermentation culture.

9. The method of claim 6, wherein the substrate in the fermentation medium is selected from one or more of the following: wheat straw, oat bran, wheat bran, rice straw shell, rice straw liquid, rice bran, defatted rice bran, rice bran oil, corn straw, corn cob husk, corn flour, corn starch, corn flour residue, corn kernel, wood, bagasse, white corn flour, white corn kernel, dried beet, soybean protein isolate powder, bran fiber powder, potato starch, rice, sterilized rice, white dextrin, sterilized white dextrin, wheat flour, sterilized wheat flour, hydroxypropyl methylcellulose, microcrystalline cellulose, xylo-oligosaccharide, lignin, cellulose, hemicellulose, cellobiose, white granulated sugar, glucose, glycerol, white vinegar, sterilized white vinegar, white spirit, ethanol solution, baking powder, or secondary processing products obtained by processing corncobs, corn husks, corn stalks, sorghum stalks, wheat stalks, rice straw stalks, bagasse, manioc waste or rice bran.

10. The method according to claim 6, wherein the substrate is present in the fermentation medium in an amount of 10 to 100 g/L.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hydrogen-producing bacterium and a method for producing hydrogen by using the same.

Background

Biological hydrogen production is one of important ways for continuously obtaining hydrogen from nature, and hydrogen-producing microorganisms are mainly divided into four types, namely green algae, cyanobacteria, photosynthetic bacteria and fermentation type bacteria, wherein the green algae, the cyanobacteria and the photosynthetic bacteria can decompose water or organic matters to obtain the hydrogen under the condition of illumination, and the fermentation type bacteria can decompose substrates to obtain the hydrogen under the condition of no illumination. At present, most of research hotspots of fermentation type hydrogen-producing bacteria concentrate on anaerobic fermentation hydrogen-producing bacteria, while facultative anaerobic fermentation hydrogen-producing bacteria or aerobic fermentation hydrogen-producing bacteria mostly have low hydrogen-producing rate and are difficult to use as industrial hydrogen-producing bacteria, for example, facultative anaerobic fermentation hydrogen-producing bacteria with high hydrogen-producing rate can be obtained, has higher tolerance to the environment, is not sensitive to illumination, oxygen or closed, and is more favorable for being used as industrial hydrogen-producing bacteria. On the other hand, the current hydrogen-producing fermentation bacteria often adopt glucose, sucrose and other sugars as substrates, the substrates are high in price and are not economical in use, and if agricultural and sideline products such as straws, corncobs, rice bran, straws and the like or secondary processing products of the agricultural and sideline products can be adopted as the substrates to produce hydrogen, the process is better in economy, and the method is favorable for realizing commercial application.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a which is preserved in China general microbiological culture Collection center (CGMCC for short) in 7 and 2 months in 2021, wherein the preservation address is No. 3 of Xilu 1 of the sunward area of Beijing, and the preservation number is CGMCC NO:22807 at the institute of microbiology of China academy of sciences.

The invention provides a hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b which is preserved in China general microbiological culture Collection center (CGMCC for short) in 7-month and 2-day 2021, wherein the preservation address is No. 3 of the West Lu No. 1 of the sunward area of Beijing, and the preservation number is CGMCC NO: 22808.

The invention also provides a method for producing hydrogen by using the hydrogen-producing bacteria, which adopts agricultural and sideline products such as straws, corncobs, rice bran, straws and the like or secondary processing products of the agricultural and sideline products as substrates to produce hydrogen.

Specifically, the present invention relates to the following aspects:

1. a hydrogen producing Bacillus (Bacillus sp.) has 16s rRNA sequence shown in SEQ ID NO. 1.

2. The hydrogen-producing Bacillus (Bacillus sp.) according to item 1, which has the number JBW1-26a and the preservation number CGMCC NO: 22807.

3. A Clostridium hydrogenogenum (Clostridium sp.) has a 16s rRNA sequence shown in SEQ ID NO. 2.

4. The Clostridium hydrogen-producing bacterium (Clostridium sp.) according to item 3, wherein the accession number is JBW1-26b and the accession number is CGMCC NO. 22808.

5. A method for producing hydrogen comprising producing hydrogen by fermentation using a hydrogen-producing microorganism, wherein the hydrogen-producing microorganism is Bacillus hydrogengenes (Bacillus sp.) JBW1-26a of item 1 or 2 and/or Clostridium hydrogengenes (Clostridium sp.) JBW1-26 of item 3 or 4; preferably the hydrogen-producing microorganisms comprise hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a and hydrogen-producing Clostridium (Clostridium sp.) JBW1-26 b; further preferably, the ratio of hydrogen producing Bacillus (Bacillus sp.) JBW1-26a to hydrogen producing Clostridium (Clostridium sp.) JBW1-26b is 0.1-10: 1.

6. The method of item 5, wherein the method comprises:

activating the hydrogen-producing microorganisms, and inoculating the activated hydrogen-producing microorganisms into a seed culture medium for culture to obtain a seed solution;

and inoculating the seed solution into a fermentation culture medium containing a substrate for fermentation culture to obtain hydrogen.

7. The method of item 6, wherein the seed medium comprises: 5.0-12.0g/L of peptone, 1-6g/L of beef extract powder, 4-6g/L of sodium chloride and 7.0-7.5 of pH value.

8. The method according to item 6, wherein the fermentation temperature is 25 to 45 ℃ and the fermentation time is 60 to 120 hours in the fermentation culture.

9. The method of item 6, wherein the substrate in the fermentation medium is selected from one or more of the following: wheat straw, oat bran, wheat bran, rice straw shell, rice straw liquid, rice bran, defatted rice bran, rice bran oil, corn straw, corn cob husk, corn flour, corn starch, corn flour residue, corn kernel, wood, bagasse, white corn flour, white corn kernel, dried beet, soybean protein isolate powder, bran fiber powder, potato starch, rice, sterilized rice, white dextrin, sterilized white dextrin, wheat flour, sterilized wheat flour, hydroxypropyl methylcellulose, microcrystalline cellulose, xylo-oligosaccharide, lignin, cellulose, hemicellulose, cellobiose, white granulated sugar, glucose, glycerol, white vinegar, sterilized white vinegar, white spirit, ethanol solution, baking powder, or secondary processing products obtained by processing corncobs, corn husks, corn stalks, sorghum stalks, wheat stalks, rice straw stalks, bagasse, manioc waste or rice bran.

10. The method according to item 6, wherein the substrate is contained in an amount of 10 to 100g/L in the fermentation medium.

Compared with the prior art, the hydrogen-producing bacterium has the following advantages: the method is insensitive to the environment, has no influence on the hydrogen production process due to the existence of light, oxygen, carbon dioxide and other gases, can utilize wide substrate sources, can use byproducts of crops or secondary processing products of the byproducts as the substrate, has high hydrogen production efficiency, is similar to anaerobic fermentation hydrogen production bacteria, and has high hydrogen content in the hydrogen production process.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

The invention provides a hydrogen-producing Bacillus (Bacillus sp). the 16s rRNA sequence of the hydrogen-producing Bacillus is shown in SEQ ID NO: 1.

1 is:

AGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTACGAGG

furthermore, the invention also provides the hydrogen-producing Bacillus (Bacillus sp.) with the number of JBW1-26a, which is preserved in China general microbiological culture Collection center (CGMCC for short) at 7.2.2021, the preservation address is No. 3 of Beijing West Lu No. 1 of the sunward Chen, and the preservation number is CGMCC NO:22807 at the institute of microbiology of China academy of sciences.

Bacillus (Bacillus sp.) is a genus of bacteria that forms spores (endospores). They have strong resistance to external harmful factors and wide distribution, and exist in soil, water, air, animal intestinal tracts and the like. Bacillus bacteria are large (4-10 μm), gram-positive, and strictly aerobic or facultative anaerobic, podded bacteria. An important property of this genus of bacteria is the ability to produce spores that are particularly resistant to adverse conditions. The genus bacillus can be divided into the following subgroups: bacillus polymyxa, Bacillus subtilis (including Bacillus cereus and Bacillus licheniformis), Bacillus brevis and Bacillus anthracis. The bacillus has the characteristics of high temperature resistance, quick revival, strong secretase and the like, and can survive under aerobic and anaerobic conditions.

The invention also provides Clostridium hydrogenogenum (Clostridium sp.) of which the 16s rRNA sequence is shown as SEQ ID NO. 2.

SEQ ID NO 2 is:

TGCAGTCGAGCGATGAAGCTCCTTCGGGAGCTGGATTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTCATAGAGGGGAATAGCCTTTCGAAAGGAAGATTAATACCGCATAAGATTGTAGTGCCGCATGGCATAGCAATTAAAGGAGTAATCCGCTATGAGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCAACGCCGCGTGAGTGATGACGGTCTTCGGATTGTAAAGCTCTGTCTTCAGGGACGATAATGACGGTACCTGAGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGCGTAAAGGGAGCGTAGGTGGATATTTAAGTGGGATGTGAAATACTCGGGCTTAACCTGGGTGCTGCATTCCAAACTGGATATCTAGAGTGCAGGAGAGGAAAGTAGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAATACCAGTGGCGAAGGCGACTTTCTGGACTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTAGGGGTTGTCATGACCTCTGTGCCGCCGCTAACGCATTAAGTATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAGACTTGACATCTCCTGAATTACCCTTAATCGGGGAAGCCCTTCGGGGCAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCTACCATTTAGTTGAGCACTCTAGCGAGACTGCCCGGGTTAACCGGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGCTGGTACAGAGAGATGCTAAACCGTGAGGTGGAGCCAAACTTTAAAACCAGTCTCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAGTTGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTGGCAAATACCCAAAGTTCTGAGCTAACGCGCAAGCGAGGCAGCGACCTAA

furthermore, the invention also provides the Clostridium (Clostridium sp.) with the number of JBW1-26b, which is preserved in China general microbiological culture Collection center (CGMCC) within 7 months and 2 days of 2021, wherein the preservation address is No. 3 of Beijing West Lu No. 1 of the sunward time, and the preservation number is CGMCC NO: 22808.

Clostridium sp is a gram-positive group of bacteria that produce spores and are highly resistant to the environment. The diameter of the spore is larger than the width of the thallus, so that the thallus is expanded into a fusiform shape, thereby obtaining the name. It is widely distributed in nature, in the soil, human and animal intestines. Is a gram-positive bacillus which can form spores and grow anaerobically. The spore is usually larger than the thallus, so the thallus is named after spindle-shaped. Also known as anaerobic bacillus. Most obligately anaerobic, few microaerophilic, most clostridia do not produce catalase, and the ability to decompose proteins and ferment sugars varies from species to species: clostridium histolyticum decomposes proteins, Clostridium perfringens ferments sugars, and Clostridium sporogenes both decomposes proteins and ferments sugars.

The present invention also provides a method for producing hydrogen, comprising producing hydrogen by fermentation using a hydrogen-producing microorganism, wherein the hydrogen-producing microorganism is the above-mentioned hydrogen-producing Bacillus (Bacillus sp.) or the above-mentioned Clostridium (Clostridium sp.), or comprises the above-mentioned hydrogen-producing Bacillus (Bacillus sp.) and the above-mentioned Clostridium (Clostridium sp.).

In a preferred embodiment, the hydrogen-producing microorganisms include Bacillus hydrogenogenicus (Bacillus sp.) JBW1-26a and Clostridium hydrogenogenicum (Clostridium sp.) JBW1-26 a. Wherein, the ratio of the hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a to the hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b can be any value.

In a particular embodiment, the ratio of hydrogen producing Bacillus (Bacillus sp.) JBW1-26a to hydrogen producing Clostridium (Clostridium sp.) JBW1-26b is 0.1-10:1, for example 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, preferably 0.5-2: 1.

The invention provides a method for producing hydrogen, which comprises the following steps:

activating the hydrogen-producing microorganisms, and inoculating the activated hydrogen-producing microorganisms into a seed culture medium for culture to obtain a seed solution;

and inoculating the seed solution into a fermentation culture medium containing a substrate for fermentation culture to obtain hydrogen.

Wherein, in a specific embodiment, the seed medium comprises: 5.0-12.0g/L of peptone, 1-6g/L of beef extract powder, 4-6g/L of sodium chloride and 7.0-7.5 of pH value.

In a specific embodiment, the seed medium comprises: 10.0g/L of peptone, 3.0g/L of beef extract powder, 5.0g/L of sodium chloride and 7.0-7.4 of pH value.

It is worth mentioning that the most common culture medium of seed culture medium is only given in the description, and the addition of glucose, starch, buffer solution, trace elements and other components beneficial to the production of bacteria to the culture medium is also applicable to the culture process of the bacteria.

Wherein the fermentation medium consists of a substrate and water.

Various substrates can be suitable for the method for producing hydrogen, and besides the components such as starch, glucose, cane sugar and the like which are usually used for producing hydrogen by fermentation, agricultural and sideline products such as straws, corncobs, rice bran, straws and the like or secondary processing products of the agricultural and sideline products can be selected as the substrates. The substrates have wide sources and better economy, and are beneficial to realizing the commercial application of the hydrogen production method.

In a specific embodiment, the substrate is selected from one or two or more of the following: wheat straw, oat bran, wheat bran, rice straw shell, rice straw liquid, rice bran, defatted rice bran, rice bran oil, corn straw, corncob, corn bran, corn flour, corn starch, corn flour residue, corn kernel, wood, bagasse, white corn flour, white corn kernel, dried beet, soybean protein isolate powder, bran fiber powder, potato starch, rice, sterilized rice, white dextrin, sterilized white dextrin, wheat flour, sterilized wheat flour, hydroxypropyl methyl cellulose, microcrystalline cellulose, xylo-oligosaccharide, lignin, cellulose, hemicellulose, cellobiose, white granulated sugar, glucose, glycerol, white vinegar, sterilized white vinegar, white spirit, ethanol solution, baking powder, or secondary processing products obtained by processing corncobs, corn husks, corn stalks, sorghum stalks, wheat stalks, rice straw stalks, bagasse, manioc waste or rice bran.

Wherein, the processing steps can adopt the processing method commonly used in the field for corncobs, corn husks, corn straws, sorghum straws, wheat straws, rice straw stalks, bagasse, cassava dregs and rice bran. For example, the processing step may include pretreatment, hydrolysis or enzymatic hydrolysis.

The pre-treatment may comprise steam explosion. Hydrolysis may include acid or base pH adjustment, 100-. The enzymolysis may comprise adding cellulase, diastase, and wooden carbohydrase to perform enzymolysis on corncob, corn husk, corn stalk, sorghum stalk, wheat stalk, rice straw, bagasse, cassava residue, and rice bran.

In a specific embodiment, the processing step is steam explosion.

In one embodiment, the processing step is acid or base pH adjustment, 100-.

In a specific embodiment, the processing step is enzymatic hydrolysis.

In one embodiment, the processing step is steam explosion followed by acid or base pH adjustment and hydrolysis at 180 deg.C.

In one embodiment, the processing step is steam explosion followed by enzymatic hydrolysis.

In a specific embodiment, the processing step is steam explosion, followed by acid or alkali pH adjustment, 100-180 ℃ high temperature hydrolysis, and finally enzymolysis.

The various substrates may, among other things, range in size, shape and processing means commonly used in the art. For example, wheat straw may include wheat straw fractions and wheat straw fractions (hydrolyzed). Wherein the wheat straw sections refer to the wheat which is cut into different lengths, and the wheat straw sections (hydrolysis) refer to the products obtained after the wheat straw is hydrolyzed.

In a specific embodiment, the wheat straw has a size of 0.5cm to 1.5cm per length.

In a particular embodiment, the wheat straw fraction (hydrolysis) is obtained by: soaking wheat straw sections in a 0.3% dilute sulfuric acid solution in a mass ratio of 8%, placing the wheat straw sections into a stainless steel reaction kettle, heating the reaction kettle to 160 ℃, maintaining the temperature for 50 minutes, cooling and taking out the wheat straw sections, and adding Ca (OH)₂Regulating the pH value of the solution to 7.0;

similarly, crushed rice straw refers to rice straw that is cut into different sizes. Crushed straw (autoclaved) refers to the product obtained by autoclaving crushed straw. The straw solution is an aqueous solution obtained by soaking straw.

In a specific embodiment, the broken straw is straw stalk cut into 0.5-1 cm.

In a particular embodiment, ground straw (autoclaved) is obtained by: putting the crushed straws into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining the pressure for 20 minutes, and then cooling and taking out the straws.

In a particular embodiment, rice hulls (autoclaved) are obtained by: putting the rice husks into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining the pressure for 20 minutes, and then cooling and taking out.

In a specific embodiment, the straw solution is an aqueous solution obtained by soaking straw in water for 4 hours and then filtering the straw.

In a particular embodiment, the straw (after washing with water) is obtained by: soaking soaked rice straw in water, cleaning, filtering, adding filtered water, dripping into iodine solution, and observing whether color change occurs, or repeating soaking and cleaning until color change does not occur.

In a particular embodiment, the rice bran (autoclaved) is obtained by: putting rice bran into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining for 20 minutes, cooling and taking out.

In a specific embodiment, the crushed corn stalks are corn stalks cut into the grain size of 0.5-2 cm.

In a particular embodiment, the ground corn stover (hydrolyzed) is obtained by: putting the crushed corn stalks into a reaction kettle, heating the reaction kettle to 120 ℃, maintaining for 30 minutes, cooling and taking out.

The corn cob is the corn cob which is threshed and is left and cut into blocks. For example, the particle size of the cut block is 0.5 to 2 cm.

In a particular embodiment, the corncobs (autoclaved) are obtained by: and putting the corncobs into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining for 20 minutes, and cooling and taking out.

The corn husk refers to corn bract. The corn bran (enzymolysis treatment) refers to a product obtained after the corn bran is subjected to enzymolysis.

In a particular embodiment, the corn bran (enzymatic treatment) is obtained by: the corn bran is put into 0.5 percent dilute sulfuric acid solution according to the mass ratio of 6 percent, put into a stainless steel reaction kettle, heated to 140 ℃ and maintained for 20 minutes, cooled and taken out, the PH value is adjusted to 5.0 by NaOH solution, and saccharifying enzyme and xylanase are added for enzymolysis for 40 hours at 50 ℃.

The white corn flour residue is a product obtained by grinding corn seeds of a white corn plant, and the white corn seeds are corn seeds of the white corn plant.

In a specific embodiment, the white corn grit is in the form of particles having a diameter of less than 0.5 mm.

Wood (chip) refers to wood chips obtained by extruding poplar wood.

In a particular embodiment, the wood (chip) is wood chips with a diameter of less than 1 mm.

Bagasse (sugar-free water washing) refers to bagasse obtained by squeezing sugarcane juice, washing with water for multiple times, and filtering until the solution is sugar-free.

Bagasse (sugar-free autoclaved) is obtained by further autoclaving bagasse (sugar-free water-washed).

In a particular embodiment, sugar cane bagasse (sugar-free autoclaved) is obtained by: the method comprises the steps of extruding sugarcane juice by sugarcane, washing and filtering for many times until the solution is free of sugar, putting the solution into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining the pressure for 30 minutes, and then cooling and taking out.

Dried beet refers to beet obtained by natural air drying.

In a specific embodiment, the content of the substrate in the fermentation medium is 10-100 g/L, for example, 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100 g/L.

In the fermentation culture, the fermentation temperature is 25-45 ℃ and the fermentation time is 60-120 h.

The generated hydrogen can be externally connected with an air bag or an inverted water-sealed glass bottle, and when the air bag is expanded or gas exists in the water-sealed glass bottle, the content of the hydrogen is detected by adopting gas chromatography.

Examples

EXAMPLE 1 screening of strains

Samples were taken from the soil in Murilian county, Liangshan, Sichuan province.

Diluting and coating the collected soil sample on a solid culture medium flat plate, and culturing for 3-5 days at 30 ℃ to obtain a primary screening strain of the multi-colony. Wherein the solid culture medium is nutrient agar culture medium, has pH value (25 deg.C) of 7.3 + -0.1, and comprises peptone 10.0g/L, beef extract powder 3.0g/L, sodium chloride 5.0g/L, and agar 15.0 g/L.

And respectively inoculating the primary screened strains into a seed culture medium, and performing shake culture at 25 ℃ for 60 hours. Taking nutrient broth as a culture medium, namely: the pH value (25 ℃) is 7.3 +/-0.1, and the pH value comprises 10.0g/L of peptone, 3.0g/L of beef extract powder and 5.0g/L of sodium chloride. And (4) sterilizing the culture medium at high pressure and high temperature after the culture medium is dissolved, and obtaining the seed culture medium after the temperature is reduced to room temperature. Then mixing the cultured seed culture medium, sterilized water and a substrate to be used as a fermentation culture medium, sealing, connecting an air bag or an inverted water-sealed glass bottle externally, and pumping gas into a gas chromatograph to judge whether hydrogen exists or not and determine the content of the hydrogen when the air bag swells or gas exists in the water-sealed glass bottle.

Wherein the used fermentation culture substrate is corn starch, and the mass ratio of the seed culture medium, the sterilized water and the substrate is 10: 85: 5.

the chromatographic conditions for hydrogen determination are shown in table 1:

TABLE 1

Diluting a fermentation culture medium with hydrogen output as a liquid sample, coating the liquid sample on a solid culture medium flat plate, taking bacterial colonies to culture in a seed culture medium, and repeating the steps.

After 12 rounds of solid medium plate screening and 300 batches of bacterial liquid hydrogen production experiments, two strains are obtained. One strain is cultured under the aerobic atmosphere, and the obtained strain is named as hydrogen producing Bacillus (Bacillus sp.) JBW1-26 a. The other strain is obtained by placing a culture medium plate into a vertical culture bag containing an anaerobic gas generating bag, and the obtained strain is named Clostridium hydrogenogenum (Clostridium sp.) JBW1-26 b.

EXAMPLE 2 identification of the strains

Identification of Bacillus (Bacillus sp.) JBW1-26a

16s rRNA of the strain was extracted for sequencing.

The sequence of the 16s rRNA obtained by determination is shown in SEQ ID NO: 1:

AGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTACGAGG(SEQ ID NO:1)。

identification of Clostridium (Clostridium sp.) JBW1-26 b:

16s rRNA of the strain was extracted for sequencing.

The sequence of the 16s rRNA obtained by determination is shown in SEQ ID NO: 2:

TGCAGTCGAGCGATGAAGCTCCTTCGGGAGCTGGATTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTCATAGAGGGGAATAGCCTTTCGAAAGGAAGATTAATACCGCATAAGATTGTAGTGCCGCATGGCATAGCAATTAAAGGAGTAATCCGCTATGAGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCAACGCCGCGTGAGTGATGACGGTCTTCGGATTGTAAAGCTCTGTCTTCAGGGACGATAATGACGGTACCTGAGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGCGTAAAGGGAGCGTAGGTGGATATTTAAGTGGGATGTGAAATACTCGGGCTTAACCTGGGTGCTGCATTCCAAACTGGATATCTAGAGTGCAGGAGAGGAAAGTAGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAATACCAGTGGCGAAGGCGACTTTCTGGACTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTAGGGGTTGTCATGACCTCTGTGCCGCCGCTAACGCATTAAGTATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAGACTTGACATCTCCTGAATTACCCTTAATCGGGGAAGCCCTTCGGGGCAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCTACCATTTAGTTGAGCACTCTAGCGAGACTGCCCGGGTTAACCGGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGCTGGTACAGAGAGATGCTAAACCGTGAGGTGGAGCCAAACTTTAAAACCAGTCTCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCTGGAGTTGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTGGCAAATACCCAAAGTTCTGAGCTAACGCGCAAGCGAGGCAGCGACCTAA(SEQ ID NO:2)

example 3 production of Hydrogen-Effect of different Strain ratios

Activated hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a and hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b are respectively inoculated into a seed culture medium for culture. Culturing for 10h at 37 ℃ in an aerobic atmosphere to obtain JBW1-26a seed solution of hydrogen-producing Bacillus (Bacillus sp.); placing into a vertical culture bag containing an anaerobic gas generating bag, and culturing at 37 deg.C for 10 hr to obtain seed solution of Clostridium hydrogenogenum (Clostridium sp.) JBW1-26 b.

Taking nutrient broth as a culture medium, namely: the pH value (25 ℃) is 7.3 +/-0.1, and the pH value comprises 10.0g/L of peptone, 3.0g/L of beef extract powder and 5.0g/L of sodium chloride. And (4) sterilizing the culture medium at high pressure and high temperature after the culture medium is dissolved, and obtaining the seed culture medium after the temperature is reduced to room temperature.

The seed solution was inoculated into a seed-containing medium at a volume ratio of 10% (v/v) of the total seed solution to the seed medium according to the composition shown in Table 2, and shake-cultured at 25 ℃ for 60 hours. Then mixing the cultured seed culture medium, sterilized water and a substrate to be used as a fermentation culture medium, sealing, connecting an air bag or an inverted water-sealed glass bottle externally, and pumping gas into a gas chromatograph to judge whether hydrogen exists or not and determine the content of the hydrogen when the air bag swells or gas exists in the water-sealed glass bottle.

Wherein the used fermentation medium is 5g of corn starch as a substrate, and the mass ratio of the seed culture medium, the sterilized water and the substrate is 10: 85: 5.

TABLE 2

The results of the hydrogen gas measurement are shown in Table 3, from which it can be seen that the effect when Bacillus JBW1-26a and Clostridium JBW1-26b are used in combination is superior to that when both bacteria are used alone, particularly that when Bacillus JBW1-26a and Clostridium JBW1-26b are used in combination at 1:1, the hydrogen-producing effect is the best.

TABLE 3

Test number	Volume of Hydrogen gas produced (ml)
		2-1	36.8
2-2	43.4
		2-3	65.6
2-4	45
		2-5	34.2

Example 4 production of Hydrogen-Hydrogen production of different substrates

Activated hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a and hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b are respectively inoculated into a seed culture medium for culture. Culturing for 10h at 37 ℃ in an aerobic atmosphere to obtain JBW1-26a seed solution of hydrogen-producing Bacillus (Bacillus sp.); placing into a vertical culture bag containing an anaerobic gas generating bag, and culturing at 37 deg.C for 10 hr to obtain seed solution of Clostridium hydrogenogenum (Clostridium sp.) JBW1-26 b.

Taking nutrient broth as a culture medium, namely: the pH value (25 ℃) is 7.3 +/-0.1, and the pH value comprises 10.0g/L of peptone, 3.0g/L of beef extract powder and 5.0g/L of sodium chloride. And (4) sterilizing the culture medium at high pressure and high temperature after the culture medium is dissolved, and obtaining the seed culture medium after the temperature is reduced to room temperature.

Inoculating the seed solution of hydrogen-producing Bacillus (Bacillus sp.) JBW1-26a and the seed solution of hydrogen-producing Clostridium (Clostridium sp.) JBW1-26b into the seed culture medium according to the volume ratio of 1:1 and the volume ratio of the total seed solution to the fermentation culture medium of 10% (v/v), and performing shake culture at 25 ℃ for 60 h. Then mixing the cultured seed culture medium, sterilized water and a substrate, sealing, externally connecting an air bag or an inverted water-sealed glass bottle, and pumping the gas into a gas chromatograph to judge whether hydrogen exists or not and determine the hydrogen content when the air bag is expanded or gas exists in the water-sealed glass bottle.

The types of the substrates of the fermentation medium are shown in Table 4, and the mass ratio of the seed culture medium, the sterilized water and the substrates is 10: 85: 5

The measurement results of hydrogen gas are shown in table 4.

Among these, the substrates used in table 4 are as follows:

the wheat straw segment cutting means that the wheat straw is cut into the segments with the length of 0.5 cm-1.5 cm;

the wheat straw segment (hydrolysis) is prepared by soaking wheat straw segment in 8% by mass in 0.3% dilute sulfuric acid solution, placing into a stainless steel reaction kettle, heating to 160 deg.C, maintaining for 50 min, cooling, taking out, adding Ca (OH)₂Regulating the pH value of the solution to 7.0;

the broken straws refer to straw straws cut into 0.5-1 cm;

ground straw (autoclaved) was obtained by: putting the crushed straws into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining the pressure for 20 minutes, and then cooling and taking out the straws;

rice hulls (autoclaved) were obtained by: putting the rice husks into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining the pressure for 20 minutes, cooling and taking out;

the straw solution is prepared by soaking straw in water for 4 hr, and filtering.

Straw (after washing) was obtained in the following manner: soaking soaked rice straw in water, cleaning, filtering, adding filtered water, dripping into iodine solution, and observing whether color change occurs, or repeating soaking and cleaning until color change does not occur.

Rice bran (autoclaved) was obtained by: putting rice bran into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining for 20 minutes, cooling and taking out.

The crushed corn stalks are cut into corn stalks with the grain size of 0.5-2 cm;

crushed corn stalks (hydrolyzed) are obtained by: putting the crushed corn stalks into a reaction kettle, heating the reaction kettle to 120 ℃, maintaining for 30 minutes, cooling and taking out.

The corncob is an irregular small block with the size of 0.5 cm-1.5 cm and is cut into blocks after the corn cob is threshed.

Corncobs (autoclaved) were obtained by: the corn cob is put into an autoclave, the autoclave is heated to 0.1-0.2 Mpa and kept for 20 minutes, and then the temperature is reduced and the corn cob is taken out.

The corn husk refers to corn bract.

The corn bran (enzymolysis treatment) is obtained by putting the corn bran into 0.5% dilute sulfuric acid solution according to the mass ratio of 6%, putting the corn bran into a stainless steel reaction kettle, heating the reaction kettle to 140 ℃ and maintaining for 20 minutes, cooling and taking out, adjusting the pH value to 5.0 by using NaOH solution, adding saccharifying enzyme and xylanase, and carrying out enzymolysis for 40 hours at 50 ℃.

The white corn flour residue is particles with the diameter less than 0.5mm of the ground corn seeds of the white corn plants.

White corn seed refers to the corn seed of the white corn plant.

Wood (shredded) is the extrusion of poplar wood into wood chips with a diameter of less than 1 mm.

Bagasse (sugar-free water wash) was obtained by: the sugarcane is squeezed to obtain sugarcane juice, and then washed with water for multiple times and filtered until the solution is free of sugar.

Bagasse (sugar-free autoclaved) was obtained by: and extruding sugarcane juice by sugarcane, washing with water for multiple times, filtering until the solution is free of sugar, putting the solution into an autoclave, heating the autoclave to 0.1-0.2 Mpa, maintaining for 30 minutes, and cooling and taking out.

Dried beet refers to beet obtained by natural air drying.

TABLE 4

Wherein, the total amount of generated hydrogen is the generated gas volume multiplied by the generated hydrogen concentration.

SEQUENCE LISTING

<110> Beijing North China science and technology Co., Ltd

<120> hydrogen-producing bacterium and method for producing hydrogen using the same

<130> TPE01616

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1454

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized

<400> 1

agtcgagcga atggattaag agcttgctct tatgaagtta gcggcggacg ggtgagtaac 60

acgtgggtaa cctgcccata agactgggat aactccggga aaccggggct aataccggat 120

aacattttga accgcatggt tcgaaattga aaggcggctt cggctgtcac ttatggatgg 180

acccgcgtcg cattagctag ttggtgaggt aacggctcac caaggcaacg atgcgtagcc 240

gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300

cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360

tgaaggcttt cgggtcgtaa aactctgttg ttagggaaga acaagtgcta gttgaataag 420

ctggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480

taatacgtag gtggcaagcg ttatccggaa ttattgggcg taaagcgcgc gcaggtggtt 540

tcttaagtct gatgtgaaag cccacggctc aaccgtggag ggtcattgga aactgggaga 600

cttgagtgca gaagaggaaa gtggaattcc atgtgtagcg gtgaaatgcg tagagatatg 660

gaggaacacc agtggcgaag gcgactttct ggtctgtaac tgacactgag gcgcgaaagc 720

gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780

tgttagaggg tttccgccct ttagtgctga agttaacgca ttaagcactc cgcctgggga 840

gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900

tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaacc 960

ctagagatag ggcttctcct tcgggagcag agtgacaggt ggtgcatggt tgtcgtcagc 1020

tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1080

atcattaagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140

acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca 1200

aagagctgca agaccgcgag gtggagctaa tctcataaaa ccgttctcag ttcggattgt 1260

aggctgcaac tcgcctacat gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320

gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380

cgaagtcggt ggggtaacct tttggagcca gccgcctaag gtgggacaga tgattggggt 1440

gaagtcgtac gagg 1454

<210> 2

<211> 1383

<212> DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized

<400> 2

tgcagtcgag cgatgaagct ccttcgggag ctggattagc ggcggacggg tgagtaacac 60

gtgggtaacc tgcctcatag aggggaatag cctttcgaaa ggaagattaa taccgcataa 120

gattgtagtg ccgcatggca tagcaattaa aggagtaatc cgctatgaga tggacccgcg 180

tcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta gccgacctga 240

gagggtgatc ggccacattg ggactgagac acggcccaga ctcctacggg aggcagcagt 300

ggggaatatt gcacaatggg ggaaaccctg atgcagcaac gccgcgtgag tgatgacggt 360

cttcggattg taaagctctg tcttcaggga cgataatgac ggtacctgag aggaagccac 420

ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggatttac 480

tgggcgtaaa gggagcgtag gtggatattt aagtgggatg tgaaatactc gggcttaacc 540

tgggtgctgc attccaaact ggatatctag agtgcaggag aggaaagtag aattcctagt 600

gtagcggtga aatgcgtaga gattaggaag aataccagtg gcgaaggcga ctttctggac 660

tgtaactgac actgaggctc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 720

ccacgccgta aacgatgaat actaggtgta ggggttgtca tgacctctgt gccgccgcta 780

acgcattaag tattccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac 840

gggggcccgc acaagcagcg gagcatgtgg tttaattcga agcaacgcga agaaccttac 900

ctagacttga catctcctga attaccctta atcggggaag cccttcgggg caggaagaca 960

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1020

cgcaaccctt attgttagtt gctaccattt agttgagcac tctagcgaga ctgcccgggt 1080

taaccgggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgtc tagggctaca 1140

cacgtgctac aatggctggt acagagagat gctaaaccgt gaggtggagc caaactttaa 1200

aaccagtctc agttcggatt gtaggctgaa actcgcctac atgaagctgg agttgctagt 1260

aatcgcgaat cagaatgtcg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320

caccatgaga gttggcaaat acccaaagtt ctgagctaac gcgcaagcga ggcagcgacc 1380

taa 1383