CN1648235A - Self floculation hydrogen producing bacteria and its sieving method - Google Patents
Self floculation hydrogen producing bacteria and its sieving method Download PDFInfo
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- CN1648235A CN1648235A CN 200410043836 CN200410043836A CN1648235A CN 1648235 A CN1648235 A CN 1648235A CN 200410043836 CN200410043836 CN 200410043836 CN 200410043836 A CN200410043836 A CN 200410043836A CN 1648235 A CN1648235 A CN 1648235A
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Abstract
The present invention relates to self flocculating hydrogen producing bacterium and its screening method and belongs to the field of the screening of new hydrogen producing bacterium. In order to raise the hydrogen producing capacity of system, the self flocculating hydrogen producing bacterium with high hydrogen producing capacity, strong acid resistance and growth superiority is separated for electron application. The self flocculating hydrogen producing bacterium is named as Ethanologen bacterium Harbin W-1 and has the preservation number of CGMCC1151. The screening process of the self flocculating hydrogen producing bacterium features the use of new culture medium. By means of self flocculation during vibrating culture, the thallus forms micro flocculent of 0.5-1.0 mm diameter and the culture medium is clear.
Description
Technical field:
The present invention relates to a kind of hydrogen-producing bacteria novel species and 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 to increase running cost at present, 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 object of the present invention is to provide a kind of hydrogen-producing bacteria (preservation date: 2004.5.25 with autoflocculation ability, high hydrogen production potential and strong acidproof ability, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center, be called for short: CGMCC, deposit number: CGMCC No.1151.) and screening method, the autoflocculation hydrogen-producing bacteria, called after Harbin producing and ethanol bacillus W-1 (EthanologenbacteriumHarbin W-1), deposit number is CGMCC1151.The screening method of autoflocculation hydrogen-producing bacteria, it adopts conventional separation, purifying and screening process, and the composition of the substratum that uses is in screening process: 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; Above-mentioned trace element is made up of following compositions: MnSO
4.7H
2O 0.01g, ZnSO
4.7H
2O0.05g, 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) 2 0.01g, constant volume 1000mL; Said 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.(its sample has sent China Committee for Culture Collection of Microorganisms common micro-organisms center to preserve to hydrogen-producing bacteria novel species Ethanologenbacterium Harbin W-1 of the present invention, preserve number is CGMCC No.1151), be from Continuous Flow biological hydrogen production reactor CSTR, to take out certain volume production hydrogen fermentation activity mud, substratum by autonomous design, adopt improved Hungate anaerobism technical point from acquisition, it is the bacterium of a strain strong flocculation ability of existing hydrogen generation efficiency efficiently and acidproof feature, this strain bacterium passes through self-flocculation when shaking culture, the little flocs unit of thalline shape, diameter 0.5～1.0mm, substratum is clarified fully, and flco is fine and close very difficult broken.It helps practical engineering application, and very big promotional value is arranged.Harbin producing and ethanol bacillus W-1 (Ethanologenbacterium Harbin W-1) biology characteristics are as follows:
1, the morphological specificity of bacterial strain:
Have very strong autoflocculation effect during this bacterial strain liquid medium within shaking culture, can form big floc particle, see Fig. 1.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～6 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.
2, the physiological and biochemical property of bacterial strain:
The growth of bacterial strain W-1 is with OD
600Absorbance represent that growth Dai Shiwei 3.8h 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.
The terminal tunning of bacterial strain W-1 liquid phase when being carbon source with glucose is mainly ethanol and acetate also has a spot of propionic acid and butyric acid, is typical ethanol-type fermentation bacterium.Reach maximum OD in cultivation 28h artifact amount
600Be 1.19.After this, cause the bacterium intraspecific competition because nutritive substance and meta-bolites become the growth limitation sex factor, bacterium enters decline phase, and bacterial metabolism is active low, and the terminal tunning of liquid phase increases mild.Cultivate behind the 48h content of ethanol and acetate and reach maximum and be respectively 3396.9mg/L and 3009.6mg/L, account for 98.6% of total volatile acid and pure content.
The fermentation gas of this bacterial strain is mainly hydrogen and carbonic acid gas, by the gas chromatographic analysis hydrogen content, calculates the hydrogen content that adds up to produce in the gas and is the accumulative total hydrogen output.The accumulative total hydrogen output increasess slowly after cultivating 36h, and the high specific hydrogen-producing speed is 25.3mmolH
2/ g drycellh, cumulative maximum hydrogen output are the 1917mL/L nutrient solution.This bacterial strain belongs to the highly effective hydrogen yield bacterium, has good energy for growth and acidproof ability, can grow under pH3.5～8.5 conditions.This strain bacterium provides valuable starting strain for realizing the biological hydrogen production industrialization, and bio-hydrogen production technology is realized that suitability for industrialized production has great theory significance and economic worth.
3, the molecular biology identification result of bacterial strain:
The 16S rRNA gene order of bacterial strain W-1 is AY434721 at the number of registration of GenBank, and the similarity of the most close kind Mierocrystalline cellulose clostridium with it (Clostridium Cellulosi) is 94%, is a new genus bacterium.16S-23S rRNA transcribed spacer sequence number of registration is AY337017, and the sequence alignment result shows that conservative region only is tRN
AlaAnd tRNA
IleSequence, the variation zone does not have the homology zone with other kind bacterial 16 S-23S rRNA transcribed spacer sequence.
Under taxonomy " genuss " be one newly to belong to, called after producing and ethanol Bacillaceae (Ethanologenbacteriumssp.) is for the taxonomy of naming first newly belongs to.It is characterized in that this genus bacterium is the obligate anaerobic growth, gram-positive microorganism; Bacillus; Peritrichous; No gemma; Metabolic characteristics is an ethanol fermentation; The similarity of Mierocrystalline cellulose clostridium (Clostridium Cellulosi) by the most close kind fusobacterium with it of 16S rRNA gene order compare of analysis is 94%.
Description of drawings: Fig. 1 is autoflocculation hydrogen-producing bacteria CGMCC1151 electron microscope morphological structure figure, and Fig. 2 forms big floc particle synoptic diagram under the autoflocculation hydrogen-producing bacteria CGMCC1151 electron microscope.
Preservation date: 2004.5.25, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center, be called for short: CGMCC, deposit number: CGMCC No.1151.
Embodiment:
The autoflocculation hydrogen-producing bacteria, called after Harbin producing and ethanol bacillus W-1 (EthanologenbacteriumHarbin W-1), deposit number is CGMCC1151.
The screening method of autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus W-1, it adopts conventional separation, purifying and screening process.
One, the separation of bacterial strain, purifying and screening: adopt the bacterium isolation medium of improvement to carry out separating of hydrogen-producing bacteria.From Continuous Flow biological hydrogen production reactor CSTR, take out 1～2mL and produce hydrogen fermentation activity mud, 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, carried out doubling dilution, be inoculated in the solid medium with sterilized water, make and roll pipe, cultivate 7～10d.Picking list bacterium colony is transferred in the liquid bulk substratum.Whether observation has floc particle to produce, and what have that flco forms is the autoflocculation bacterium.Repeat above operation several times, in good condition until flocculation, being confirmed to be with Electronic Speculum denys to be pure bacterial strain.The flocculation fermenting bacteria of separation and purification is transferred in liquid nutrient medium, and 36 ℃, 120r/min shaking culture 3d takes a sample from its gas phase with the 1mL asepsis injector, and whether detect has hydrogen to exist in its gas phase composition.If be autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus W-1.
Substratum: 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;
Above-mentioned trace element 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
2O0.2g, AlK (SO
4)
20.01g, constant volume 1000mL;
Said 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.
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 '-AGAGTITGATCCTGGCTCAG-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 '.Correspond respectively to 189～209 bases of 1486～1505 and the 23S rRNA of E.coli16S rRNA.(50 μ L) is as follows for the PCR reaction system: 10 * buffer (mg
2+) 5 μ L, 2.5mmol/LdNTP 5 μ L, 20pmol/L primer each 1 μ L, ExTaq DNA enzyme 0.25 μ L.Pcr amplification condition: 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 fermentation and hydrogen production technology: 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 the parameters such as product hydrogen temperature, pH value of zymotechnique.
Technological condition for fermentation when utilizing autoflocculation hydrogen-producing bacteria CGMCC1151 to produce hydrogen: 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.
Annex:
Sequence table
Attached one: the 16S rRNA sequence of autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus W-1 (EthanologenbacteriumHarbin W-1) (the GenBank number of registration is AY434721):
＜110〉Harbin Institute of Technology
＜120〉autoflocculation hydrogen-producing bacteria and screening method thereof
<160>2
<210>1
<211>1495
<212>RNA
＜213〉producing and ethanol Bacillaceae (Ethanologenbacterium ssp.)
<400>1
agagtttgat?cctggctcag?gacgaacgct?ggcggcgcgc?ctaacacatg?caagtcgagc 60
ggagtccttc?gggacttagc?ggcggacggg?tgagtaacgc?gtgagcaacc?tggccttcag 120
agggggataa?cgtctggaaa?cggacgctaa?taccgcatga?catggcggag?tcgcatggct 180
ctgccatcaa?aggagtaatc?cgctgaggga?tgggctcgcg?tccgattagg?tagttggtga 240
ggtaacggct?caccaagccc?gcgatcggta?gccggactga?gaggttggcc?ggccacattg 300
ggactgagac?acggcccaga?ctcctacggg?aggcagcagt?gggggatatt?gcacaatgga 360
ggaaactctg?atgcagcgac?gccgcgtgag?ggaagaaggt?cttcggattg?taaacctctg 420
tctttgggga?cgaatcaatg?acggtaccca?aggaggaagc?cacggctaac?tacgtgccag 480
cagccgcggt?aatacgtagg?tggcaagcgt?tgtccggaat?tactgggtgt?aaagggtgcg 540
caggcggggc?ggcaagttgg?atgtgaaaac?tccgggctca?acccggagcc?tgcattcaaa 600
actgtcgctc?ttgagtaagt?agaggcaggc?ggaattcccg?gtgtagcggt?gaaatgcgta 660
gatatcggga?ggaacaccag?tggcgaaggc?ggcctgctgg?gcttttactg?acgctgaggc 720
acgaaagcat?gggtagcaaa?caggattaga?taccctggta?gtccatgccg?taaacgatga 780
ttgctaggtg?tggggggtct?gaccccttcc?gtgccggagt?taacacaata?agcaatccac 840
ctggggagta?cggccgcaag?gttgaaactc?aaaggaattg?acgggggccc?gcacaagcag 900
tggagtatgt?ggtttaattc?gaagcaacgc?gaagaacctt?accaggtctt?gacatccacc 960
gaatccccca?gagatggggg?agtgtccttc?ggggagcggt?gagacaggtg?gtgcatggtt 1020
gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc?aacgagcgca?acccttgtga 1080
atagttgcta?cgaaagagca?ctctattcag?accgccgttg?acaaaacgga?ggaaggtggg 1140
gatgacgtca?aatcatcatg?ccccttatga?cctgggctac?acacgtacta?caatggccat 1200
caacagaggg?aagcaaggcc?gcgaggtgga?gcgaacccct?aaaaatggtc?tcagttcaga 1260
ttgcaggctg?aaacccgcct?gcatgaagat?ggaattgcta?gtaatcgcgg?atcagcatgc 1320
cgcggtgaat?acgttcccgg?gccttgtaca?caccgcccgt?cacaccatga?gagccgggga 1380
cacccgaagt?cggttgggta?accgtaagga?gcccgccgcc?gaaggtggaa?tcggtaattg 1440
gggtgaagtc?gtaacaaggt?agccgtatcg?gaaggtgcgg?ctagatcacc?tcctt 1495
The 16S-23S rRNA transcribed spacer sequence (the GenBank number of registration is AY337017) of Annex II autoflocculation hydrogen-producing bacteria Harbin producing and ethanol bacillus W-1 (EthanologenbacteriumHarbinW-1):
<210>2
<211>1217
<212>RNA
＜213〉producing and ethanol Bacillaceae (Ethanologenbacterium ssp.)
<400>2
aattcgagct?cggtacccgg?ggatcctcta?gagattaaag?atgttgttta?attttgaggg 60
tccgggcgga?cgcggttcgc?ttttaagccg?ccggccatgc?agatcggatt?gggagcgaac 120
agccccgatg?caggaagcat?gacggcagtt?tgaagacggg?cggcgtccca?aagaaccttc 180
aaacggtgga?aaagagaata?ggacgaccga?ttcccaggca?aagcgtgggg?tatagctttc 240
aggctgagca?gtccgagaac?aggagatttt?cctgaacttg?cgccgcgcag?cggtgaactt 300
gccgctacgg?tgagtagggg?gtgtagctca?gctgggagag?cacctgcttt?gcacgcaggg 360
ggtcaggagt?tcgattctcc?tcatctccac?cacgcctgcg?gcggatatga?cgcgccaagg 420
cagccgaaaa?cgaaaatgca?aagccgcgag?gccaagcgtc?gacgttgagg?aagccaaagc 480
atgcatgccg?gttgcaggca?aaccaaatgg?cccgggcggg?aacggacgac?cgagatgctc 540
ggattgcatg?ggctcatagc?tcaggtggtt?agagcgtgcg?cctgataagc?gcaaggtcgg 600
tggttcgagt?ccacttgagc?ccaccagcgg?gcagtgcccg?cacacaattc?gcgggaagtt 660
tgttcaaagc?agaggtcggc?cgccgcgaaa?gcgagacgga?tcgaagcaag?aacagatgga 720
tcgcaaaacc?gcttgcaaaa?gaggtaagat?ttcaggctga?gcagccgcgt?aagcaacgac 780
tttcctgaac?ttgcctccgc?cgcaggcggg?gggaccttga?aaactgaata?atgttctaat 840
ctgcaatttt?tttaatgaac?gaagcgagta?gcggcggaag?tttcaggctg?agcagctgtg 900
taagcagtgg?gcttgcttga?acatacgtct?tgcgaagcaa?gttcgttggg?taaaattccg 960
attgacacat?aaggtcaagc?tacaaagagc?gcaaggggaa?tgccttggca?ccaggagccg 1020
atgaaggacg?cagcgatctg?cgaaaagcca?cggggagtcg?aaagcaggca?ttgatccgtg 1080
gatatccgaa?tggggcaacc?cggcggagcc?atactccgtc?agcgtgcagt?gaatccatag 1140
ctgcacgtgg?ggaaccgcct?gaactgaaac?atctaagtac?caatcgtcga?cctgcaggca 1200
tgcaagcttg?gcactgg 1217
aattcgagct?cggtacccgg?ggatcctcta?gagattaaag?atgttgttta?attttgaggg 60
tccgggcgga?cgcggttcgc?ttttaagccg?ccggccatgc?agatcggatt?gggagcgaac 120
agccccgatg?caggaagcat?gacggcagtt?tgaagacggg?cggcgtccca?aagaaccttc 180
aaacggtgga?aaagagaata?ggacgaccga?ttcccaggca?aagcgtgggg?tatagctttc 240
aggctgagca?gtccgagaac?aggagatttt?cctgaacttg?cgccgcgcag?cggtgaactt 300
gccgctacgg?tgagtagggg?gtgtagctca?gctgggagag?cacctgcttt?gcacgcaggg 360
ggtcaggagt?tcgattctcc?tcatctccac?cacgcctgcg?gcggatatga?cgcgccaagg 420
cagccgaaaa?cgaaaatgca?aagccgcgag?gccaagcgtc?gacgttgagg?aagccaaagc 480
atgcatgccg?gttgcaggca?aaccaaatgg?cccgggcggg?aacggacgac?cgagatgctc 540
ggattgcatg?ggctcatagc?tcaggtggtt?agagcgtgcg?cctgataagc?gcaaggtcgg 600
tggttcgagt?ccacttgagc?ccaccagcgg?gcagtgcccg?cacacaattc?gcgggaagtt 660
tgttcaaagc?agaggtcggc?cgccgcgaaa?gcgagacgga?tcgaagcaag?aacagatgga 720
tcgcaaaacc?gcttgcaaaa?gaggtaagat?ttcaggctga?gcagccgcgt?aagcaacgac 780
tttcctgaac?ttgcctccgc?cgcaggcggg?gggaccttga?aaactgaata?atgttctaat 840
ctgcaatttt?tttaatgaac?gaagcgagta?gcggcggaag?tttcaggctg?agcagctgtg 900
taagcagtgg?gcttgcttga?acatacgtct?tgcgaagcaa?gttcgttggg?taaaattccg 960
attgacacat?aaggtcaagc?tacaaagagc?gcaaggggaa?tgccttggca?ccaggagccg 1020
atgaaggacg?cagcgatctg?cgaaaagcca?cggggagtcg?aaagcaggca?ttgatccgtg 1080
gatatccgaa?tggggcaacc?cggcggagcc?atactccgtc?agcgtgcagt?gaatccatag 1140
ctgcacgtgg?ggaaccgcct?gaactgaaac?atctaagtac?caatcgtcga?cctgcaggca 1200
tgcaagcttg?gcactgg 1217
Claims (4)
1. a strain autoflocculation hydrogen-producing bacteria, called after Harbin producing and ethanol bacillus W-1 (Ethanologenbacterium Harbin W-1), deposit number is CGMCC1151.
2. autoflocculation hydrogen-producing bacteria according to claim 1 is characterized in that the GenBank number of registration of the 16S rRNA sequence of ethanol bacillus W-1 is AY434721, and 16S rRNA sequence is:
agagtttgat?cctggctcag?gacgaacgct?ggcggcgcgc?ctaacacatg?caagtcgagc 60
ggagtccttc?gggacttagc?ggcggacggg?tgagtaacgc?gtgagcaacc?tggccttcag 120
agggggataa?cgtctggaaa?cggacgctaa?taccgcatga?catggcggag?tcgcatggct 180
ctgccatcaa?aggagtaatc?cgctgaggga?tgggctcgcg?tccgattagg?tagttggtga 240
ggtaacggct?caccaagccc?gcgatcggta?gccggactga?gaggttggcc?ggccacattg 300
ggactgagac?acggcccaga?ctcctacggg?aggcagcagt?gggggatatt?gcacaatgga 360
ggaaactctg?atgcagcgac?gccgcgtgag?ggaagaaggt?cttcggattg?taaacctctg 420
tctttgggga?cgaatcaatg?acggtaccca?aggaggaagc?cacggctaac?tacgtgccag 480
cagccgcggt?aatacgtagg?tggcaagcgt?tgtccggaat?tactgggtgt?aaagggtgcg 540
caggcggggc?ggcaagttgg?atgtgaaaac?tccgggctca?acccggagcc?tgcattcaaa 600
actgtcgctc?ttgagtaagt?agaggcaggc?ggaattcccg?gtgtagcggt?gaaatgcgta 660
gatatcggga?ggaacaccag?tggcgaaggc?ggcctgctgg?gcttttactg?acgctgaggc 720
acgaaagcat?gggtagcaaa?caggattaga?taccctggta?gtccatgccg?taaacgatga 780
ttgctaggtg?tggggggtct?gaccccttcc?gtgccggagt?taacacaata?agcaatccac 840
ctggggagta?cggccgcaag?gttgaaactc?aaaggaattg?acgggggccc?gcacaagcag 900
tggagtatgt?ggtttaattc?gaagcaacgc?gaagaacctt?accaggtctt?gacatccacc 960
gaatccccca?gagatggggg?agtgtccttc?ggggagcggt?gagacaggtg?gtgcatggtt 1020
gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc?aacgagcgca?acccttgtga 1080
atagttgcta?cgaaagagca?ctctattcag?accgccgttg?acaaaacgga?ggaaggtggg 1140
gatgacgtca?aatcatcatg?ccccttatga?cctgggctac?acacgtacta?caatggccat 1200
caacagaggg?aagcaaggcc?gcgaggtgga?gcgaacccct?aaaaatggtc?tcagttcaga 1260
ttgcaggctg?aaacccgcct?gcatgaagat?ggaattgcta?gtaatcgcgg?atcagcatgc 1320
cgcggtgaat?acgttcccgg?gccttgtaca?caccgcccgt?cacaccatga?gagccgggga 1380
cacccgaagt?cggttgggta?accgtaagga?gcccgccgcc?gaaggtggaa?tcggtaattg 1440
gggtgaagtc?gtaacaaggt?agccgtatcg?gaaggtgcgg?ctagatcacc?tcctt 1495。
3. autoflocculation hydrogen-producing bacteria according to claim 1 is characterized in that the GenBank number of registration of the 16S-23S rRNA transcribed spacer sequence of Harbin producing and ethanol bacillus W-1 is AY337017, and 16S-23SrRNA transcribed spacer sequence is:
aattcgagct?cggtacccgg?ggatcctcta?gagattaaag?atgttgttta?attttgaggg 60
tccgggcgga?cgcggttcgc?ttttaagccg?ccggccatgc?agatcggatt?gggagcgaac 120
agccccgatg?caggaagcat?gacggcagtt?tgaagacggg?cggcgtccca?aagaaccttc 180
aaacggtgga?aaagagaata?ggacgaccga?ttcccaggca?aagcgtgggg?tatagctttc 240
aggctgagca?gtccgagaac?aggagatttt?cctgaacttg?cgccgcgcag?cggtgaactt 300
gccgctacgg?tgagtagggg?gtgtagctca?gctgggagag?cacctgcttt?gcacgcaggg 360
ggtcaggagt?tcgattctcc?tcatctccac?cacgcctgcg?gcggatatga?cgcgccaagg 420
cagccgaaaa?cgaaaatgca?aagccgcgag?gccaagcgtc?gacgttgagg?aagccaaagc 480
atgcatgccg?gttgcaggca?aaccaaatgg?cccgggcggg?aacggacgac?cgagatgctc 540
ggattgcatg?ggctcatagc?tcaggtggtt?agagcgtgcg?cctgataagc?gcaaggtcgg 600
tggttcgagt?ccacttgagc?ccaccagcgg?gcagtgcccg?cacacaattc?gcgggaagtt 660
tgttcaaagc?agaggtcggc?cgccgcgaaa?gcgagacgga?tcgaagcaag?aacagatgga 720
tcgcaaaacc?gcttgcaaaa?gaggtaagat?ttcaggctga?gcagccgcgt?aagcaacgac 780
tttcctgaac?ttgcctccgc?cgcaggcggg?gggaccttga?aaactgaata?atgttctaat 840
ctgcaatttt?tttaatgaac?gaagcgagta?gcggcggaag?tttcaggctg?agcagctgtg 900
taagcagtgg?gcttgcttga?acatacgtct?tgcgaagcaa?gttcgttggg?taaaattccg 960
attgacacat?aaggtcaagc?tacaaagagc?gcaaggggaa?tgccttggca?ccaggagccg 1020
atgaaggacg?cagcgatctg?cgaaaagcca?cggggagtcg?aaagcaggca?ttgatccgtg 1080
gatatccgaa?tggggcaacc?cggcggagcc?atactccgtc?agcgtgcagt?gaatccatag 1140
ctgcacgtgg?ggaaccgcct?gaactgaaac?atctaagtac?caatcgtcga?cctgcaggca 1200
tgcaagcttg?gcactgg 1217。
4, a kind of screening method of autoflocculation hydrogen-producing bacteria, it adopts conventional separation, purifying and screening process, it is characterized in that the composition of the substratum that uses in screening process is: glucose 20g, Tryptones 4g, extractum carnis 2g, yeast water 1g, NaCl 4g, K
2HPO
41.5g, L-halfcystine 0.5g, FeSO
4.7H
2O0.1g, MgCl
20.1g, micro-10ml, VITAMIN 10ml, resazurin 0.2%w/v;
Above-mentioned trace element 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
2O0.2g, AlK (SO
4)
20.01g, constant volume 1000mL;
Said 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321174C (en) * | 2005-09-28 | 2007-06-13 | 哈尔滨工业大学 | Continuous flow culture method of industrial biological hydrogen preparing spawn and biological hydrogen preparing system reinforcing method |
| CN110607337A (en) * | 2019-09-24 | 2019-12-24 | 哈尔滨工业大学 | Method for producing hydrogen by mutual-culture interaction of fermented hydrogen-producing bacteria and electroactive bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321174C (en) * | 2005-09-28 | 2007-06-13 | 哈尔滨工业大学 | Continuous flow culture method of industrial biological hydrogen preparing spawn and biological hydrogen preparing system reinforcing method |
| CN110607337A (en) * | 2019-09-24 | 2019-12-24 | 哈尔滨工业大学 | Method for producing hydrogen by mutual-culture interaction of fermented hydrogen-producing bacteria and electroactive bacteria |
| CN110607337B (en) * | 2019-09-24 | 2022-08-26 | 哈尔滨工业大学 | Method for producing hydrogen by mutual-culture interaction of fermented hydrogen-producing bacteria and electroactive bacteria |
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