CN1624109A - Self-coagulating producing hydrogen bacteria and its screening process - Google Patents
Self-coagulating producing hydrogen bacteria and its screening process Download PDFInfo
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- CN1624109A CN1624109A CN 200410043835 CN200410043835A CN1624109A CN 1624109 A CN1624109 A CN 1624109A CN 200410043835 CN200410043835 CN 200410043835 CN 200410043835 A CN200410043835 A CN 200410043835A CN 1624109 A CN1624109 A CN 1624109A
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Abstract
A self-flocculating hydrogen-generating bacterium, Harbin ethanolgenic bacterium YUAN-3 (CGMCC No.1152) is disclosed, which is an obligate anaerobic bacillus with the metabolic characteristic of ethanol fermenting. Its maximal specific hydrogen generating rate is 22.6 mmlH2/g drycell.h. It can become the depositive flocculated particles in the hydrogen generating bioreactor. The hydrogen can be continuously generated without carrier and filler.
Description
Technical field:
The present invention relates to a strain fermentative hydrogen-producing bacteria and a screening method thereof, be specifically related to a strain autoflocculation fermentative hydrogen-producing bacteria novel species Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium Harbin YUAN-3) and a screening method thereof.
Background technology:
Bio-hydrogen production technology is one of important means that solves energy dilemma and problem of environmental pollution, because it has double effects and sustainability characteristics, is subjected to the attention of countries in the world day by day.Improving hydrogen generation efficiency is the difficult problem that the bio-hydrogen production technology industrialization needs to be resolved hurrily.Filtering out the highly effective hydrogen yield bacterium is to accelerate to realize the industrialized effective way of biological hydrogen production.In order to obtain the highly effective hydrogen yield fermenting bacteria, the foreign study person has separated a large amount of product hydrogen fermenting bacterias, and isolating product hydrogen fermentation strain majority concentrates on fusobacterium and enterobacter.Kumar etc. are separated to enterobacter cloacae (Enterobacter cloacae) IIT-BT08 from the leaf extract, this bacterial strain is at 36 ℃, and under medium pH value 6.0 conditions, maximum hydrogen-producing speed is 29.63mmol H
2/ g dry cellh is the highest bacterium of reporting at present of hydrogen production potential.The Continuous Flow zymotechnique of hydrogen-producing bacteria is one of key link of fermentation method bio-hydrogen production technology realization suitability for industrialized production.Utilize the autoflocculation bacterium to adopt filling bed type reactor (PBR) that enteroaerogen (Enterobacter aerogenes) HU-101 and AY-2 have been carried out the 18d cultured continuously as (1998) such as Rachman.Kumar etc. (2000) with coir fixedly enterobacter cloacae (Enterobacter clocae) IIT-BT08 carried out the Continuous Flow cultivation.Mainly be to adopt immobilization technology at present, not only increased running cost, and be difficult for keeping biomass.Therefore separating the autoflocculation bacterium takes the hydrogen-producing bacteria of on-fixedization to have more practical significance.Because the growth conditions of hydrogen-manufacturing reactor is different with static cultivation, especially the pH of reactive system is all lower in the acidogenic fermentation process, though therefore at present isolating efficient fermenting bacteria have higher hydrogen production potential, but in reactor, often can not reach its best hydrogen and growth conditions of producing, the highly effective hydrogen yield bacterium can not become the dominant population in the microflora, can only take immobilization way, limit it and produced hydrogen usefulness, increase running cost.For the hydrogen production potential of further raising system, need separate both highly effective hydrogen yield and strong acid resistance, can become the autoflocculation bacterium of dominant growth again, have more realistic meaning and engineering using value.
Summary of the invention:
The purpose of this invention is to provide a kind of autoflocculation hydrogen-producing bacteria and screening method thereof with autoflocculation ability, high hydrogen production potential and strong acidproof ability, it helps practical engineering application, has very big promotional value.Autoflocculation hydrogen-producing bacteria of the present invention Harbin producing and ethanol bacillus YUAN-3 is in (address: China, China Committee for Culture Collection of Microorganisms common micro-organisms center, Beijing, the Zhong Guan-cun, postcode: 100080) preservation, called after Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium HarbinYUAN-3), deposit number is CGMCC No.1152, and preservation date is on May 25th, 2004.Harbin producing and ethanol bacillus YUAN-3 of the present invention takes out certain volume production hydrogen fermentation activity mud from Continuous Flow biological hydrogen production reactor CSTR, substratum by autonomous design, adopt improved Hungate anaerobism technical point from what obtain, it is the bacterium that the strong flocculation ability of the existing hydrogen generation efficiency efficiently of a strain has acidproof feature again.The screening method of Harbin producing and ethanol bacillus YUAN-3 of the present invention is as follows: (one) adopts the bacterium isolation medium bacterial strain to be carried out separation, purifying and the screening of hydrogen-producing bacteria; (2), carry out the preferred of autoflocculation highly effective hydrogen yield bacterial strain with the autoflocculation hydrogenogens strain repeated isolation, the purifying that filter out.The bacterial strain of gained of the present invention is the bacterium novel species, and affiliated taxonomy " genus " is one newly to belong to, and called after producing and ethanol Bacillaceae (Ethanologenbacteriumssp.) is for the taxonomy of name first newly belongs to.Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium Harbin YUAN-3) has following biological property:
One, morphological specificity: bacterium is a Gram-positive bacillus, and pod membrane is arranged, no endogenous spore, and it is long that obligate is detested health, bacterium length changeable (wide 0.4 μ m～0.5 μ m * length 2 μ m～8 μ m).The thalline peritrichous is found in transmission electron microscope observation, and it is uneven that the scanning electron microscope observation thalline has the surface to present.When on solid medium, growing, be the circular bacterium colony of oyster white, smooth surface, neat in edge.Have very strong autoflocculation effect during this bacterial strain liquid medium within shaking culture, can form big floc particle.
Two, physiological and biochemical property: the growth of bacterial strain YUAN-3 is with OD
600Absorbance represent that growth Dai Shiwei 4.0h is calculated in the growth by quantity in the bacterium logarithm period unit.Can utilize the citric acid growth, cow's milk litmus test, M.R., V.P., gelatin are tested all positive, nitrate test and H
2The S test all is negative.With molasses and glucose is substrate, and the suitableeest fermentation and hydrogen production and autoflocculation temperature are 36 ℃, and the suitableeest growth pH is 4.5, and the suitableeest product hydrogen pH is 4.0.Bacterial strain YUAN-3 main tunning when being carbon source with glucose is ethanol, acetate, H
2, CO
2With a spot of lactic acid, the percentage composition of ethanol and acetate accounts for more than 90% of volatile acid and pure total amount, and ethanol content is not less than 45% of volatile acid and pure total amount.The high specific hydrogen-producing speed of bacterial strain YUAN-3 is 22.6mmolH
2/ g drycellh, cumulative maximum hydrogen output are the 1568mL/L nutrient solution.
Three: the molecular biology identification result of bacterial strain: the 16S rRNA gene order of bacterial strain YUAN-3 is AY675965 at the number of registration of GenBank, the similarity of the most close kind Mierocrystalline cellulose clostridium with it (ClostridiumCellulosi) is 94%, is a new genus bacterium.16S-23S rRNA transcribed spacer sequence number of registration is AY556390, and the sequence alignment result shows that conservative region only is tRNA
AlaAnd tRNA
IleSequence, the variation zone does not have the homology zone with other kind bacterial 16 S-23S rRNA transcribed spacer sequence.
Four: the engineering application characteristic: autoflocculation hydrogen-producing bacteria bacterial strain YUAN-3 of the present invention can utilize carbohydrate such as soybean, corn, potato and molasses containing waste water and beer waste water to produce hydrogen.Because it has the autoflocculation characteristic, therefore need not add filler or carrier, be added to the hydrogen of can miscarrying continuously behind Continuous Flow stirred-tank reactor and the biological expanded bed reactor.Volumetric loading 60～70kgCOD/ (m
3D) time, produce hydrogen digit rate average out to 6～10L/ (L reactor d).The bacterium that filters out according to method of the present invention not only has higher hydrogen production potential, can form good floc particle in reactor, keeps very big biomass, reaches its best hydrogen and growth conditions of producing, and makes it become dominant population in the microflora.Owing to have stronger autoflocculation ability, do not need carrier when therefore fermenting, reduced running cost, have realistic meaning and engineering using value.
Description of drawings:
Fig. 1 is an autoflocculation hydrogen-producing bacteria of the present invention Harbin producing and ethanol bacillus YUAN-3 autoflocculation design sketch, Fig. 2 is the sem photograph of autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus YUAN-3, and Fig. 3 is the transmission electron microscope picture of autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus YUAN-3.
Embodiment:
Embodiment one: the Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium Harbin YUAN-3) of present embodiment carries out seed selection according to following step:
One, the separation of bacterial strain, purifying and screening: adopt the bacterium isolation medium of improvement to carry out separating of hydrogen-producing bacteria, its concrete steps are as follows: a, take out 1～2mL produce hydrogen fermentation activity mud from Continuous Flow biological hydrogen production reactor CSTR, put into the triangular flask that is full of nitrogen, add several granulated glass spherees, 1h vibrates on shaking table, zoogloea in the mud is smashed, be diluted to 10 with sterilized water
-1～10
-11Ml; B, with 0.2～0.5ml inoculation in the 10ml solid medium, make and roll pipe, cultivate 7～10d; C, picking list bacterium colony are transferred in the liquid nutrient medium, and whether observation has floc particle to produce, and what have that flco forms is the autoflocculation bacterium; D, the above operation of repetition 3～10 times, in good condition until flocculation, the particle diameter that promptly flocculates is 0.5～1.5cm, and substratum is clarified fully, and flco still can flocculate after smashing again, and being confirmed to be with Electronic Speculum is not pure bacterial strain; E, the flocculation fermenting bacteria of separation and purification is transferred in liquid nutrient medium, shaking culture 3d under 36 ℃, the condition of 120r/min, taking a sample from its gas phase with the 1mL asepsis injector, whether in its gas phase composition have hydrogen exist, if be the autoflocculation hydrogen-producing bacteria if detecting.Described substratum (solid medium) is made up of following compositions: glucose 10～30g, Tryptones 2～6g, extractum carnis 1～4g, yeast water 0.1～1.5g, NaCl 2～6g, K
2HPO
40.5～3g, L-halfcystine 0.1～1g, FeSO
4.7H
2O 0.1～0.5g, MgCl
20.1～0.5g, trace element 5～20ml, VITAMIN 5～15ml, resazurin 0.01～0.1% (w/v).Described liquid nutrient medium is made up of following compositions: glucose 10～30g, Tryptones 2～6g, extractum carnis 1～4g, yeast water 0.1～1.5g, NaCl 2～6g, K
2HPO
40.5～3g, L-halfcystine 0.1～1g, FeSO
4.7H
2O 0.1～0.5g, MgCl
20.1～0.5g, trace element 5～20ml, VITAMIN 5～15ml, resazurin 0.01～0.1% (w/v), agar 1～2% (w/v).Described substratum (solid medium) preferably is made up of following compositions: glucose 20g, Tryptones 4g, extractum carnis 2g, yeast water 1g, NaCl 4g, K
2HPO
41.5g, L-halfcystine 0.5g, FeSO
4.7H
2O 0.1g, MgCl
20.1g, micro-10ml, VITAMIN 10ml, resazurin 0.02% (w/v).Described liquid nutrient medium preferably is made up of following compositions: glucose 20g, Tryptones 4g, extractum carnis 2g, yeast water 1g, NaCl 4g, K
2HPO
41.5g, L-halfcystine 0.5g, FeSO
4.7H
2O 0.1g, MgCl
20.1g, micro-10ml, VITAMIN 10ml, resazurin 0.02% (w/v), agar 1～2% (w/v).Described trace element is made up of following compositions: MnSO
4.7H
2O0.01～0.05g, ZnSO
4.7H
2O 0.05～0.1g, H
3BO
30.01～0.05, N (CH
2COOH)
34～5g, CaCl
2.2H
2O 0.01～0.05g, Na
2MoO
40.01～0.05g, CoCl
2.6H
2O 0.1～0.5g, AlK (SO
4)
20.01～0.05g, constant volume 1000mL; Described VITAMIN is made up of following compositions: cobalamin 0.01～0.05g, xitix 0.01～0.05g, riboflavin 0.01～0.05g, citric acid 0.01～0.05g, Vitamin B6 0.03～0.1g, folic acid 0.01～0.05g, para-amino benzoic acid 0.01～0.05g, creatine 0.01～0.05g, constant volume 1000mL.Described trace element preferably is made up of following compositions: MnSO
4.7H
2O 0.01g, ZnSO
4.7H
2O 0.05g, H
3BO
30.01, N (CH
2COOH)
34.5g, CaCl
2.2H
2O 0.01g, Na
2MoO
40.01g, CoCl
2.6H
2O 0.2g, AlK (SO
4)
20.01g, constant volume 1000mL; Described VITAMIN preferably is made up of following compositions: cobalamin 0.01g, xitix 0.025g, riboflavin 0.025g, citric acid 0.02g, Vitamin B6 0.05g, folic acid 0.01g, para-amino benzoic acid 0.01g, creatine 0.025g, constant volume 1000mL.
Two, autoflocculation highly effective hydrogen yield bacterial strain is preferred: with the autoflocculation hydrogenogens strain that filters out, repeated isolation, purification process, after treating that its aerogenesis is stable, survey its hydrogen production potential, and the bacterial strain that hydrogen production potential is high, repeated isolation operation is taken at and produces the bigger bacterial colony of bubble in the solid medium and carry out purifying, above-mentioned steps repeatedly, bacterium is stable in rolling pipe produces till the big and many bubble; After the enrichment, survey its hydrogen production potential again, repeated multiple times is till optimizing the highest bacterial strain of strong autoflocculation and hydrogen production potential.
Three, the physiological and biochemical analysis of bacterium: the growth ultraviolet spectrophotometer of bacterium is at the absorbance of 600nm place working sample, turbid as bacterium.It is that sample is centrifugal under 5000r/min that the turbid bacterium dry weight of different bacterium is measured, and abandons supernatant liquor, washes bacterial precipitation 2 times with sterile pure, and 80 ℃ of dry 12h claim its weight with the precise electronic balance.Morphology evaluation reference literature method (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 2001.).
Four, strain classification is identified: extract bacterial genomes DNA, being used for 16S rDNA amplification PCR reaction primer is universal primer: forward primer BSF8/20:5 '-AGAGTTTGATCCTGGCTCAG-3 '; Reverse primer BSR1541/1522:5 '-ACGGCTACCTTGTTACGACT-3 ' corresponds respectively to 1522～1541 bases of 8～27 and the 16S rRNA of colon bacillus (E.coli) 16S rRNA.The amplimer of 16S-23S rRNAITS is PSA1486/1505:5 '-GGGGTGAAGTCGTAACAAGG-3 '; PLA209/189:5 '-GGTACTTAGATGT TTCAGTTC-3 ' corresponds respectively to 189～209 bases of 1486～1505 and the 23S rRNA of E.coli16S rRNA.(50 μ L) is as follows for described PCR reaction system: 10 * buffer (mg
2+) 5 μ L, 2.5mmol/L dNTP 5 μ L, 20pmol/L primer each 1 μ L, ExTaq DNA enzyme 0.25 μ L.Described pcr amplification condition is as follows: 95 ℃ of 5min; 94 ℃ of 1min; 60 ℃ of 30S; 72 ℃ of 1min, 30 circulations; 72 ℃ of 8min use the pMD18-T carrier cloning, carry out sequencing.
Five, hydrogen-producing bacteria tunning and best fermentation and hydrogen production condition analysis: the analysis volatile acid of tunning and alcohols are measured, and use GC122 type gas chromatograph, get the 1mL nutrient solution, centrifugal 5000r/min gets the supernatant liquor sample introduction, column length 2m, carrier GDX103,60～80 orders, hydrogen flame detector, nitrogen is done carrier gas, flow velocity 60mL/min, hydrogen flow rate are 50mL/min, and air velocity is 490mL/min, 210 ℃ of vaporizing chambers, 190 ℃ of post and sensing chamber.Hydrogen and CO 2 measuring use SC-II type gas chromatograph, and column length 2m, carrier are TDS-01,60～80 orders, and thermal conductivity cell detector, nitrogen is done carrier gas, and flow velocity is 70mL/min, 150 ℃ of post and sensing chamber.By the static test optimization for fermentation technology, determine to influence the suitableeest substrate, optimum growh and product hydrogen temperature, the pH value of zymotechnique.The bacterial strain YUAN-3 that present embodiment filters out has following feature:
One, morphological specificity: bacterium is a Gram-positive bacillus, and pod membrane is arranged, no endogenous spore, and it is long that obligate is detested health, bacterium length changeable (wide 0.4 μ m～0.5 μ m * length 2 μ m～8 μ m).The thalline peritrichous is found in transmission electron microscope observation, and it is uneven that the scanning electron microscope observation thalline has the surface to present.When on solid medium, growing, be the circular bacterium colony of oyster white, smooth surface, neat in edge.Have very strong autoflocculation effect during this bacterial strain liquid medium within shaking culture, can form big floc particle, diameter 0.5～1.5cm, substratum is clarified fully, and floc particle still can flocculate again after smashing and (see Fig. 1～Fig. 3).
Two, physiological and biochemical property: the growth of bacterial strain YUAN-3 is with OD
600Absorbance represent that growth Dai Shiwei 4.0h is calculated in the growth by quantity in the bacterium logarithm period unit.Can utilize the citric acid growth, cow's milk litmus test, M.R., V.P., gelatin are tested all positive, nitrate test and H
2The S test all is negative.With molasses and glucose is substrate, and the suitableeest fermentation and hydrogen production and autoflocculation temperature are 36 ℃, and the suitableeest growth pH is 4.5, and the suitableeest product hydrogen pH is 4.0.Bacterial strain YUAN-3 main tunning when being carbon source with glucose is ethanol, acetate, H
2, CO
2With a spot of lactic acid, the percentage composition of ethanol and acetate accounts for more than 90% of volatile acid and pure total amount, and ethanol content is not less than 45% of volatile acid and pure total amount.The fermentation gas of this bacterial strain is mainly hydrogen and carbonic acid gas, and hydrogen percentage composition 45%～55%, high specific hydrogen-producing speed are 22.6mmolH
2/ g drycellh, cumulative maximum hydrogen output are the 1568mL/L nutrient solution.
Three, the molecular biology identification result of bacterial strain: the 16S rRNA gene order of bacterial strain YUAN-3 is AY675965 at the number of registration of GenBank, the similarity of the most close kind Mierocrystalline cellulose clostridium with it (ClostridiumCellulosi) is 94%, is a new genus bacterium.16S-23S rRNA transcribed spacer sequence number of registration is AY556390, and the sequence alignment result shows that conservative region only is tRNAAAla and tRNAIle sequence, and the variation zone does not have the homology zone with other kind bacterial 16 S-23S rRNA transcribed spacer sequence.
Sequence table
One, the 16S rRNA sequence of autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium HarbinYUAN-3) (the GenBank number of registration is AY675965):
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAGCGGAGTCCTTCGGGACTTAGCGGCGGACGGGTGAGTAACGCGTGAGCAACCTGGCCTTCAGAGGGGGATAACGTCTGGAAACGGACGCTAATACCGCATGACATGGCGGAGTCGCATGGCTCTGCCATCAAAGGAGTAATCCGCTGAGGGATGGGCTCGCGTCCGATTAGGTAGTTGGTGAGGTAACGGCTCACCAAGCCCGCGATCGGTAGCCGGACTGAGAGGTTGGCCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAGGGAAGAAGGTCTTCGGATTGTAAACCTCTGTCTTTGGGGACGAATCAATGACGGTACCCAAGGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTACTGGGTGTAAAGGGTGCGCAGGCGGGGCGGCAAGTTGGATGTGAAAACTCCGGGCTCAACCCGGAGCCTGCATTCAAAACTGTCGCTCTTGAGTGAAGTAGAGGCAGGCGGAATTCCCGGTGTAGCGGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCGAAGGCGGCCTGCTGGGCTTTTACTGACGCTGAGGCACGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTGCTAGGTGTGGGGGGTCTGACCCCTTCCGTGCCGGAGTTAACACAATAAGCAATCCACCTGGGGAGTACGGGTCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCTCGCACAAGCAGTGGAGTATGTGGTTTGATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCACCGAATCCCCCAGAGATTGGGGAGTGCCCTTCGGGGAGCGGTGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTGAATAGTTGCTACGAAAGAGCACTCTATTCAGACCGCCGTTGACAAAACGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCATCAACAGAGGGAAGCAAGGCCGCGAGGTGGAGCGAACCCCTAAAAATGGTCTCAGCTCAGATTGCAGGCTGAAACCCGCCTGCATGAAGATGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGCCGGGGACACCCGAAGTCGGTTGGGTAACCGTAAGGAGCCCGCCGCCGAAGGTGGAATCGGTAATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTA
Two, the 16S-23S rRNA transcribed spacer sequence (the GenBank number of registration is AY556390) of autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium HarbinYUAN-3)
GGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGAGACTCGTTTCTTTAGAAACAGTGTCAGCGCAGGGATTTGTGGAAAGATGTTGTTTAATTTTGAGGGTCCGGGCGGACGCGGTTCGCTTTTAAGCCGCCGGCCATGCAGATCGGATTGGGAGCGAACAGCCCCGATGCAGGAAGCATGACGGCAGTTTGAAGACGGGCGGCGTCCCAAAGAACCTTCAAACGGTGGAAAAGAGAATAGGACGACCGATTCCCAGGCAAAGCGTGGGGTATAGCTTTCAGGCTGAGCAGTCCGAGAACAGGAGATTTTCCTGAACTTGCGCCGCGCAGCGGTGAACTTGCCGCTACGGTGAGTAGGGGGCGTAGCTCAGCTGGGAGAGCACCTGCTTTGCACGCAGGGGGTCAGGAGTTCGATTCTCCTCATCTCCACCACGCCTGCGGCGGATATGACGCGCCAAGGCAGCCGAAAACGAAAATGCAAAGCCGCGAGGCCAAGCGTCGACGTTGAGGAAGCCAAAGCATGCATGCCGGTTGCAGGCAAACCAAATGGCCCGGGCGGGAACGGACGACCGAGATGCTCGGATTGCATGGGCTCATAGCTCAGGTGGTTAGAGCGTGCGCCTGATAAGCGCAAGGTCGGTGGTTCGAGTCCACTTGAGCCCACCAGCGGGCAGTGCCCGCACACAATTCGCGGGAAGTTTGTTCAAAGCAGAGGTCGGCCGCCGCGAAAGCGAGACGGATCGAAGCAAGAACAGATGGATCGCAAAACCGCTTGCAAAAGAGGTAAGATTTCAGGCTGAGCAGCCGCGTAAGCAACGACTTTCCTGAACTTGCCTCCGCCGCAGGCGGGGGGACCTTGAAAACTGAATAATGTTCTAATCTGCAATTTTTTTAATGAACGAAGCGAGTAGCGGCGGAAGTTTCAGGTTGAGCAGCTGTGTAAGCAGTGGGCTTGCTTGAACATACGTCTTGCGAAGCAAGTTCGTTGGGTAAAATTCCGATTGACACATAAGGTCAAGCTACAAAGAGCGCAAGGGGAATGCCTTGGCACCAGGAGCCGATGAAGGACGCAGCGATCTGCGAAAAGCCACGGGGAGTCGAAAGCAGGCATTGATCCGTGGATATCCGAATGGGGCAACCCGGCGGAGCCATACTCCGTCAGCGTGCAGTGAATCCATAGCTGCACGTGGGGAACCGCCTGAACTGAAACATCTAAGTACC
Claims (9)
1, autoflocculation hydrogen-producing bacteria, called after Harbin producing and ethanol bacillus (EthanologenbacteriumHarbin YUAN-3), deposit number is CGMCC No.1152.
2, autoflocculation hydrogen-producing bacteria according to claim 1 is characterized in that the GenBank number of registration of the 16S rRNA sequence of described Harbin producing and ethanol bacillus YUAN-3 is AY675965, and 16S rRNA sequence is:
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAGCGGAGTCCTTCGGGACTTAGCGGCGGACGGGTGAGTAACGCGTGAGCAACCTGGCCTTCAGAGGGGGATAACGTCTGGAAACGGACGCTAATACCGCATGACATGGCGGAGTCGCATGGCTCTGCCATCAAAGGAGTAATCCGCTGAGGGATGGGCTCGCGTCCGATTAGGTAGTTGGTGAGGTAACGGCTCACCAAGCCCGCGATCGGTAGCCGGACTGAGAGGTTGGCCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAGGGAAGAAGGTCTTCGGATTGTAAACCTCTGTCTTTGGGGACGAATCAATGACGGTACCCAAGGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTACTGGGTGTAAAGGGTGCGCAGGCGGGGCGGCAAGTTGGATGTGAAAACTCCGGGCTCAACCCGGAGCCTGCATTCAAAACTGTCGCTCTTGAGTGAAGTAGAGGCAGGCGGAATTCCCGGTGTAGCGGTGAAATGCGTAGATATCGGGAGGAACACCAGTGGCGAAGGCGGCCTGCTGGGCTTTTACTGACGCTGAGGCACGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTGCTAGGTGTGGGGGGTCTGACCCCTTCCGTGCCGGAGTTAACACAATAAGCAATCCACCTGGGGAGTACGGGTCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCTCGCACAAGCAGTGGAGTATGTGGTTTGATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCACCGAATCCCCCAGAGATTGGGGAGTGCCCTTCGGGGAGCGGTGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTGAATAGTTGCTACGAAAGAGCACTCTATTCAGACCGCCGTTGACAAAACGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCATCAACAGAGGGAAGCAAGGCCGCGAGGTGGAGCGAACCCCTAAAAATGGTCTCAGCTCAGATTGCAGGCTGAAACCCGCCTGCATGAAGATGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGCCGGGGACACCCGAAGTCGGTTGGGTAACCGTAAGGAGCCCGCCGCCGAAGGTGGAATCGGTAATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTA。
3, autoflocculation hydrogen-producing bacteria according to claim 1, the GenBank number of registration that it is characterized in that the 16S-23S rRNA transcribed spacer sequence of described Harbin producing and ethanol bacillus YUAN-3 (Ethanologenbacterium Harbin YUAN-3) is AY556390, and 16S-23S rRNA transcribed spacer sequence is:
GGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGAGACTCGTTTCTTTAGAAACAGTGTCAGCGCAGGGATTTGTGGAAAGATGTTGTTTAATTTTGAGGGTCCGGGCGGACGCGGTTCGCTTTTAAGCCGCCGGCCATGCAGATCGGATTGGGAGCGAACAGCCCCGATGCAGGAAGCATGACGGCAGTTTGAAGACGGGCGGCGTCCCAAAGAACCTTCAAACGGTGGAAAAGAGAATAGGACGACCGATTCCCAGGCAAAGCGTGGGGTATAGCTTTCAGGCTGAGCAGTCCGAGAACAGGAGATTTTCCTGAACTTGCGCCGCGCAGCGGTGAACTTGCCGCTACGGTGAGTAGGGGGCGTAGCTCAGCTGGGAGAGCACCTGCTTTGCACGCAGGGGGTCAGGAGTTCGATTCTCCTCATCTCCACCACGCCTGCGGCGGATATGACGCGCCAAGGCAGCCGAAAACGAAAATGCAAAGCCGCGAGGCCAAGCGTCGACGTTGAGGAAGCCAAAGCATGCATGCCGGTTGCAGGCAAACCAAATGGCCCGGGCGGGAACGGACGACCGAGATGCTCGGATTGCATGGGCTCATAGCTCAGGTGGTTAGAGCGTGCGCCTGATAAGCGCAAGGTCGGTGGTTCGAGTCCACTTGAGCCCACCAGCGGGCAGTGCCCGCACACAATTCGCGGGAAGTTTGTTCAAAGCAGAGGTCGGCCGCCGCGAAAGCGAGACGGATCGAAGCAAGAACAGATGGATCGCAAAACCGCTTGCAAAAGAGGTAAGATTTCAGGCTGAGCAGCCGCGTAAGCAACGACTTTCCTGAACTTGCCTCCGCCGCAGGCGGGGGGACCTTGAAAACTGAATAATGTTCTAATCTGCAATTTTTTTAATGAACGAAGCGAGTAGCGGCGGAAGTTTCAGGTTGAGCAGCTGTGTAAGCAGTGGGCTTGCTTGAACATACGTCTTGCGAAGCAAGTTCGTTGGGTAAAATTCCGATTGACACATAAGGTCAAGCTACAAAGAGCGCAAGGGGAATGCCTTGGCACCAGGAGCCGATGAAGGACGCAGCGATCTGCGAAAAGCCACGGGGAGTCGAAAGCAGGCATTGATCCGTGGATATCCGAATGGGGCAACCCGGCGGAGCCATACTCCGTCAGCGTGCAGTGAATCCATAGCTGCACGTGGGGAACCGCCTGAACTGAAACATCTAAGTACC。
4, the screening method of autoflocculation hydrogen-producing bacteria is characterized in that it is achieved in that one, adopts the bacterium isolation medium bacterial strain to be carried out separation, purifying and the screening of hydrogen-producing bacteria; Two,, carry out the preferred of autoflocculation highly effective hydrogen yield bacterial strain with the autoflocculation hydrogenogens strain repeated isolation, the purifying that filter out.
5, the screening method of autoflocculation hydrogen-producing bacteria according to claim 4 is characterized in that described substratum comprises following compositions: glucose, Tryptones, extractum carnis, yeast water, NaCl, K
2HPO
4, L-halfcystine, FeSO
4.7H
2O, MgCl
2, trace element, VITAMIN and resazurin.
6, the screening method of autoflocculation hydrogen-producing bacteria according to claim 4 is characterized in that described substratum (solid medium) is made up of following compositions: glucose 10～30g, Tryptones 2～6g, extractum carnis 1～4g, yeast water 0.1～1.5g, NaCl 2～6g, K
2HPO
40.5～3g, L-halfcystine 0.1～1g, FeSO
4.7H
2O 0.1～0.5g, MgCl
20.1～0.5g, trace element 5～20ml, VITAMIN 5～15ml, resazurin 0.01～0.1% (w/v).
7, the screening method of autoflocculation hydrogen-producing bacteria according to claim 4 is characterized in that the content of each composition in the described substratum is: glucose 20g, Tryptones 4g, extractum carnis 2g, yeast water 1g, NaCl 4g, K
2HPO
41.5g, L-halfcystine 0.5g, FeSO
4.7H
2O 0.1g, MgCl
20.1g, micro-10ml, VITAMIN 10ml, resazurin 0.2% (w/v).
8,, it is characterized in that described trace element is made up of following compositions: MnSO according to the screening method of claim 5,6 or 7 described autoflocculation hydrogen-producing bacterias
4.7H
2O 0.01g, ZnSO
4.7H
2O 0.05g, H
3BO
30.01, N (CH
2COOH)
34.5g, CaCl
2.2H
2O 0.01g, Na
2MoO
40.01g, CoCl
2.6H
2O0.2g, AlK (SO
4)
20.01g, constant volume 1000mL.
9, according to the screening method of claim 5,6 or 7 described autoflocculation hydrogen-producing bacterias, it is characterized in that described VITAMIN is made up of following compositions: cobalamin 0.01g, xitix 0.025g, riboflavin 0.025g, citric acid 0.02g, Vitamin B6 0.05g, folic acid 0.01g, para-amino benzoic acid 0.01g, creatine 0.025g, constant volume 1000mL.
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