CN104593285A - Enterobacter cloacae gxas-h1 and use thereof - Google Patents
Enterobacter cloacae gxas-h1 and use thereof Download PDFInfo
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- CN104593285A CN104593285A CN201410522297.8A CN201410522297A CN104593285A CN 104593285 A CN104593285 A CN 104593285A CN 201410522297 A CN201410522297 A CN 201410522297A CN 104593285 A CN104593285 A CN 104593285A
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Abstract
The invention discloses enterobacter cloacae gxas-h1 and a use thereof. The enterobacter cloacae is named as gxas-h1 and has a preservation number of CCTCC NO. M 2014366. The enterobacter cloacae gxas-h1 can be used for hydrogen fermentative-production in a medium containing cane sugar as a main raw material. The hydrogen fermentative-production method utilizing the enterobacter cloacae gxas-h1 has high hydrogen production efficiency and a low cost and can be industrialized easily.
Description
Technical field
The present invention relates to technical field of bioengineering, specifically an Enterobacter cloacae (Enterobacter cloacae) gxas-h1, obtain from the screening of suburb, Nanning collected specimens, this bacterial strain can be applicable to be that fermenting raw materials produces hydrogen with sucrose.
Background technology
Hydrogen can clean renewable, and burning only produces water and huge energy, becomes one of 21 century important fuel.The contradiction that bio-hydrogen production technology reaction conditions is gentle, energy consumption is low, can properly settle energy and environment, promotes the coordinated development of ecnomics and enviroment, again becomes the problem making earnest efforts in the world exploring and studying.
At present, the method for hydrogen manufacturing mainly comprises traditional physics and chemistry method hydrogen manufacturing and hydrogen generation by biological process.Traditional physics and chemistry method hydrogen manufacturing comprises water electrolysis hydrogen producing, mineral fuel hydrogen manufacturing, solar hydrogen making, thermochemical cycle for hydrogen production and plasma hydrogen manufacturing etc.But traditional physics and chemistry method hydrogen manufacturing has the following disadvantages: (1) mineral fuel hydrogen manufacturing has CO
2discharge, contaminate environment; (2) raw materials cost and the facility investment of mineral fuel hydrogen manufacturing, water electrolysis hydrogen producing and thermochemical cycle for hydrogen production are high; (3) plasma hydrogen manufacturing energy consumption is excessive.
Bio-hydrogen production technology comprises optical drive hydrogen manufacturing and anaerobically fermenting hydrogen manufacturing two kinds of modes.But because the former utilizes photosynthetic bacterium to convert solar energy into Hydrogen Energy, its optical energy utilization efficiency is lower, photoreactor difficult design, so be difficult at no distant date apply; And the latter is the anaerobically fermenting utilizing hydrogenogens, its substrate conversion efficiency is high, and hydrogen-producing speed is fast, and reactor design is simple, and renewable resources and organic waste can be utilized to produce, and easilier relative to the former realizes application in short-term.
At present, be in the world still in the research of bio-hydrogen production technology the laboratory study stage, be difficult to practical application.
Summary of the invention
The object of this invention is to provide an Enterobacter cloacae (Enterobacter cloacae) gxas-h1 and application thereof, this bacterial strain has good fermentation optimization and genetic modification prospect, also has important industrial application value simultaneously.
Enterobacter cloacae of the present invention (Enterobacter cloacae) gxas-h1 obtains from Nanning sampling separation screening, and its preserving number is CCTCC NO.M 2014366, and separating screening method is as follows:
(1) sample: from Nanning collected specimens;
(2) enrichment: each sample respectively takes 1g and is placed in 250mLLB substratum, is placed in 37 DEG C of incubators and cultivates 24h, gets the high karusen of gas production rate and carries out primary dcreening operation;
(3) primary dcreening operation: by karusen dilution spread high for gas production rate screening plate culture medium, be placed in 37 DEG C of incubators and cultivate 24h, select oyster white from flat board, translucent, circular, smooth, the sticking bacterium colony of neat in edge, projection carries out multiple sieve;
(4) multiple sieve: picking individual colonies is in 1% sucrose fermention medium from screening flat board, be placed in heat collecting type magnetic force heating stirrer and cultivate 37 DEG C of fermentation 24h, connect collection and confinement of gases bag, detect through gas chromatograph, the bacterial strain that screening hydrogen output is higher is optimized;
(5) identify: identify that this bacterial strain is enterobacter cloacae (Enterobacter cloacae) through 16S rDNA.
The invention provides the application of enterobacter cloacae (Enterobacter cloacae) gxas-h1 in fermentative production hydrogen.
Enterobacter cloacae provided by the present invention (Enterobacter cloacae) gxas-h1 can be used for fermentative production hydrogen, its method is: be inoculated in fermention medium by enterobacter cloacae bacterial strain (Enterobacter cloacae) gxas-h1, be placed in heat collecting type magnetic force heating stirrer and cultivate 37 DEG C of fermentation 16 ~ 24h, obtain the gas mixture containing hydrogen.
As preferably, described fermention medium take sucrose as the fermention medium of primary carbon source, and the mass percentage of sucrose is 0.5 ~ 1%.
Preferred as the best, described fermention medium take sucrose as the fermention medium of primary carbon source, and the mass percentage of sucrose is 1%.
As preferably, by mass percentage, peptone 0.1% ~ 0.2% is also comprised in described fermention medium, yeast extract 0.2% ~ 0.3%, sodium-chlor 0.2% ~ 0.3%, potassium primary phosphate 0.05% ~ 0.2%, ammonium sulfate 0.3% ~ 0.4%, magnesium sulfate heptahydrate 0.04% ~ 0.05%, surplus is distilled water.
Preferred as the best, by mass percentage, also comprise peptone 0.1% in described fermention medium, yeast extract 0.3%, sodium-chlor 0.3%, potassium primary phosphate 0.2%, ammonium sulfate 0.4%, magnesium sulfate heptahydrate 0.05%, surplus is distilled water.
As preferably, described inoculation is inoculated in fermention medium according to the inoculum size being 5 ~ 10% by the enterobacter cloacae bacterial strain growing to logarithmic phase (Enterobacter cloacae) gxas-h1.
Preferred as the best, described inoculation is inoculated in fermention medium according to the inoculum size being 10% by the enterobacter cloacae bacterial strain growing to logarithmic phase (Enterobacter cloacae) gxas-h1.
Described hydrogen detecting instrument equipment and numbering: GC-9750 gas chromatograph.Gas chromatographic detection condition is: carrier gas: high pure nitrogen, flow 10mL/min; Injector temperature 80 DEG C, post case initial temperature is 50 DEG C, keeps 5min, is warming up to 120 DEG C with the speed of 20 DEG C/min, keeps 1.5min; Detector temperature 130 DEG C; Sampling volume 500uL.The relative retention time of hydrogen is about 1.3 ~ 1.4min.
Enterobacter cloacae of the present invention (Enterobacter cloacae) gxas-h1 can be used for being that fermenting raw materials produces hydrogen with sucrose, and hydrogen output reaches as high as 155.9mL/g (sucrose).
beneficial effect of the present invention
1, the present invention separates a kind of sucrose that can utilize for the new strains of fermenting raw materials height hydrogen producing from occurring in nature, has widened the bacterial classification utilizing fermentable to produce hydrogen further.
2, the present invention utilizes the method hydrogen generation efficiency of enterobacter cloacae (Enterobacter cloacae) gxas-h1 fermentation and hydrogen production high, and cost is low, is easy to industrialization.
Accompanying drawing explanation
The colonial morphology of Fig. 1 enterobacter cloacae (Enterobacter cloacae) gxas-h1 on LB substratum
the explanation of preservation information
Enterobacter cloacae (Enterobacter cloacae) gxas-h1, its deposit number is CCTCC NO.M2014366, preservation date is on August 4th, 2014, and depositary institution is China typical culture collection center, and preservation address is wuchang, wuhan road Jia Shan Wuhan University.
Embodiment
Method in the following example, if no special instructions, is ordinary method.
Embodiment 1: the separation of enterobacter cloacae (Enterobacter cloacae) gxas-h1, selection systems
The first step: sampling
In countryside, Nanning collected specimens.
Second step: enrichment
Each sample respectively takes 1g and is placed in the triangular flask that enrichment medium is housed respectively, is placed in 30 DEG C of shaking tables, and 220r/min cultivates 72h, and the bacterial strain selecting fermentation gas higher screens.
Enrichment medium component: peptone 10g, yeast extract 5g, sodium-chlor 10g, with distilled water constant volume to 1000mL, pH 7.0.
3rd step: primary dcreening operation
By diluted sample higher for karusen viscosity coating primary dcreening operation plate culture medium, be placed in 37 DEG C of incubators and cultivate 16h, select oyster white, translucent, circular, smooth, the sticking bacterium colony of neat in edge, projection carries out multiple sieve.
Primary dcreening operation plate culture medium: peptone 10g, yeast extract 5g, sodium-chlor 10g, agar 20g, distilled water 1000mL, pH 7.0.
4th step: multiple sieve
From primary dcreening operation culture medium flat plate, picking individual colonies is in fermention medium, be placed in heat collecting type magnetic force heating stirrer and cultivate 37 DEG C of fermentation 24h, connect collection and confinement of gases bag, detect through gas chromatograph, finishing screen is selected a strain and is utilized the bacterial strain that sucrose fermentative production hydrogen output is higher, called after gxas h-1.
Sucrose fermention medium: sucrose 10g, peptone 1g, yeast extract 3g, sodium-chlor 3g, dipotassium hydrogen phosphate 2g, ammonium sulfate 4g, magnesium sulfate heptahydrate 0.5g, with distilled water constant volume to 1000mL, pH 7.0.
5th step: qualification
16S rDNA identifies:
Enterobacter cloacae (Enterobacter cloacae) gxas-h116S rDNA sequence is increased, order-checking obtains sequence for shown in SEQ ID NO.1, the sequence obtained is compared in GenBank database (www.ncbi.nlm.nih.gov) after sequencing analysis.The sequencing results shows: enterobacter cloacae bacterial strain (Enterobacter cloacae) gxas-h1 and enterobacter cloacae bacterial strain (Enterobacter cloacae) homology are more than 99%.
Identify thus, bacterial strain gxas h-1 belongs to enterobacter cloacae bacterial strain (Enterobacter cloacae).
The present invention separates a kind of new strains of the substratum fermentation high yield hydrogen that sucrose can be utilized to be raw material from occurring in nature, has widened the bacterial classification utilizing fermentable to produce hydrogen further.
Embodiment 2: enterobacter cloacae (Enterobacter cloacae) gxas-h1 fermentative production hydrogen
Get enterobacter cloacae (Enterobacter cloacae) the gxas-h1 spread plate substratum of glycerol stocks, be placed in 37 DEG C of incubators and cultivate 20h, choose single bacterium colony in seed culture medium, being cultured to OD600 at 37 DEG C of shaking speed 220r/min is about 1.0, the inoculum size being 10% according to volume percent is inoculated in the 250mL triangular flask of dress 100mL fermention medium, connect collection and confinement of gases bag, be placed in heat collecting type magnetic force heating stirrer and cultivate 37 DEG C of fermentation 20h, connect air collector and collect gas, detect through gas chromatograph, the bacterial strain that screening hydrogen output is higher is optimized, hydrogen output can reach 155.9mL/g (sucrose).
Plate culture medium: peptone 10g, yeast extract 5g, sodium-chlor 10g, agar 20g, distilled water 1000mL, pH 7.0,121 DEG C of high pressure steam sterilization 20min.
Seed culture medium: peptone 10g, yeast extract 5g, sodium-chlor 10g, with distilled water constant volume to 1000mL, pH 7.0,121 DEG C of high pressure steam sterilization 20min.
Sucrose fermention medium: sucrose 10g, peptone 1g, yeast extract 3g, sodium-chlor 3g, dipotassium hydrogen phosphate 2g, ammonium sulfate 4g, magnesium sulfate heptahydrate 0.5g, pH 7.0, with distilled water constant volume to 1000mL.
Plant and instrument and numbering: GC-9750 gas chromatograph.Gas chromatographic detection condition is: carrier gas: high pure nitrogen, flow 10mL/min; Injector temperature 80 DEG C, post case initial temperature is 50 DEG C, keeps 5min, is warming up to 120 DEG C with the speed of 20 DEG C/min, keeps 1.5min; Detector temperature 130 DEG C; Sampling volume 500uL.The relative retention time of hydrogen is about 1.3 ~ 1.4min.
Can be found out by above-described embodiment, enterobacter cloacae (Enterobacter cloacae) gxas-h1 has good fermentation and hydrogen production performance, after being optimized the fermention medium taking sucrose as primary carbon source, this bacterium fermentation and hydrogen production efficiency improves further.
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (9)
1. an Enterobacter cloacae, its Classification And Nomenclature is (Enterobacter cloacae) gxas-h1, and deposit number is CCTCC NO.M 2014366.
2. the application of enterobacter cloacae according to claim 1 in fermentative production hydrogen.
3. the application of enterobacter cloacae according to claim 1 in fermentative production hydrogen, its method is as follows: be inoculated in fermention medium by enterobacter cloacae (Enterobacter cloacae) gxas-h1, connect collection and confinement of gases bag, be placed in heat collecting type magnetic force heating stirrer, leavening temperature is 37 DEG C, and fermentation 16 ~ 24 h collect the gas mixture obtained containing hydrogen.
4. application according to claim 3, it is characterized in that, described inoculation is inoculated in fermention medium according to the inoculum size that volume percent is 5 ~ 10% by the enterobacter cloacae growing to logarithmic phase (Enterobacter cloacae) gxas-h1.
5. application according to claim 3, is characterized in that: described fermention medium take sucrose as the fermention medium of primary carbon source, and the mass percentage of sucrose is 0.5 ~ 1%.
6. application according to claim 5, is characterized in that: described fermention medium take sucrose as the fermention medium of primary carbon source, and the mass percentage of sucrose is 1%.
7. application according to claim 5, it is characterized in that: by mass percentage, also containing peptone 0.1% ~ 0.2% in described fermention medium, yeast extract 0.2% ~ 0.3%, sodium-chlor 0.2% ~ 0.3%, potassium primary phosphate 0.05% ~ 0.2%, ammonium sulfate 0.3% ~ 0.4%, magnesium sulfate heptahydrate 0.04% ~ 0.05%, surplus is distilled water.
8. application according to claim 7, is characterized in that: by mass percentage, peptone 0.1% in described fermention medium, yeast extract 0.3%, sodium-chlor 0.3%, dipotassium hydrogen phosphate 0.2%, ammonium sulfate 0.4%, magnesium sulfate heptahydrate 0.05%, surplus is distilled water.
9. application according to claim 4, is characterized in that, described inoculation is inoculated in fermention medium according to the inoculum size that volume percent is 10% by the enterobacter cloacae growing to logarithmic phase (Enterobacter cloacae) gxas-h1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400825A (en) * | 2015-12-24 | 2016-03-16 | 清华大学 | Method for improving efficiency of producing methane through kitchen waste high-concentration anaerobic digestion |
| CN106085896A (en) * | 2016-05-10 | 2016-11-09 | 清华大学深圳研究生院 | Enterobacter cloacae and application thereof |
| CN110724653A (en) * | 2019-07-04 | 2020-01-24 | 淮阴师范学院 | Enterobacter cloacae HYN-P47 and biological suspending agent prepared from same |
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| CN102226159A (en) * | 2010-11-29 | 2011-10-26 | 山东大学 | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol |
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| CN102226159A (en) * | 2010-11-29 | 2011-10-26 | 山东大学 | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400825A (en) * | 2015-12-24 | 2016-03-16 | 清华大学 | Method for improving efficiency of producing methane through kitchen waste high-concentration anaerobic digestion |
| CN106085896A (en) * | 2016-05-10 | 2016-11-09 | 清华大学深圳研究生院 | Enterobacter cloacae and application thereof |
| CN106085896B (en) * | 2016-05-10 | 2019-07-09 | 清华大学深圳研究生院 | Enterobacter cloacae and application thereof |
| CN110724653A (en) * | 2019-07-04 | 2020-01-24 | 淮阴师范学院 | Enterobacter cloacae HYN-P47 and biological suspending agent prepared from same |
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