CN105754889B - A kind of enterococcus faecium and its application in hydrogen production through anaerobic fermentation - Google Patents
A kind of enterococcus faecium and its application in hydrogen production through anaerobic fermentation Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of enterococcus faecium and its application in hydrogen production through anaerobic fermentation.The manure enterococcin strain is INET2, and classification naming is Enterococcus faecium, is CGMCC1.15321 in the deposit number of China General Microbiological culture presevation administrative center.The enterococcus faecium is isolated from through 5kGy gamma-ray irradiation treated anaerobically digested sludge, is one plant of new enterococcus faecium with hydrogen production potential.The enterococcus faecium has the characteristics that grow rapid, hydrogen generation efficiency height, drug resistance, heat-resisting, resistance to acid and alkali；Requirement of the fermentation and hydrogen production system to environment can be reduced, the economic benefit of whole system is promoted；Clean energy resource hydrogen can be made by substrate of debirs simultaneously, can achieve the double benefit of Waste disposal and production of energy.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of enterococcus faecium and its in hydrogen production through anaerobic fermentation
Using.
Background technique
With urbanization and industrialized progress, energy crisis and environmental pollution have become what the limitation mankind were further developed
Two big main problems.Fossil fuel is constantly declining as current main energy sources donor, reserves；And it is fired with fossil fuel
Environmental problem caused by the pollutant (such as carbon dioxide, sulfur dioxide, PM2.5 etc.) generated is burnt also to get worse.Hydrogen has
High heating value burns the features such as pollution-free, becomes the reasonable selection of clean energy resource.
Traditional hydrogen preparation method includes the steam recombining reaction (SRM/SRH) of methane and other hydrocarbons, is changed
The non-catalytic partial oxidation (POX) of stone fuel, in conjunction with the thermogravimetric group of SRM and POX and the cell reaction of water.However, above method
All along with a large amount of consumption of energy and to a degree of pollution of environment.Compared with above-mentioned conventional method, hydrogen generation by biological process gas
Possess more advantages: mild reaction condition, low power consuming and feature of environmental protection height etc..Biological hydrogen production includes photosynthetic organism hydrogen generation, light hair
Ferment biological hydrogen production and dark dark microbial fermentation for bio-hydrogen production, wherein dark fermentation method is compared with other bioanalysis, device structure is simpler, reacts item
Part is easier to control, and the operation is more convenient, thus receives more concerns of researchers.In addition, using debirs as secretly
The substrate of fermentation may also reach up waste processing and the regenerated dual purpose of energy recovery.
The main body of dark Hydrogen Bio-production By Anaerobic Fermentation is the production hydrogen microorganism in system, and common microbial flora includes clostridium
Belong to, such as: Clostridium butyricum, Clostridium tyrobutyricum, Clostridium
Thermocellum etc.；Enterobacter, such as Enterobacter cloacae, Enterobacter aerogenes etc.；Ai Xi
Bordetella, such as Escherichia coli；And some other kinds such as Ethanoligenens harbinese,
Ruminococcus albus, Caldicellulosiruptor saccharolyticus etc..
All the time, its drug resistance and its probiotic properties are concentrated mainly on for the research of enterococcus faecium.In view of reality
Waste, such as the complexity of municipal refuse, municipal sludge, agricultural wastes ingredient, common microorganism is often by poison therein
Object is inhibited, and the drug resistance and strong viability that enterococcus faecium has, and reduces the selectivity to substrate.Therefore, dung is utilized
Enterococcus progress fermentation and hydrogen production has the advantages that significant.Up to the present, there are no enterococcus faecium is applied to fermentation and hydrogen production
Correlative study report.
Summary of the invention
A kind of application the present invention provides enterococcus faecium and its in hydrogen production through anaerobic fermentation, specific technical solution are as follows:
A kind of enterococcus faecium, the manure enterococcin strain are INET2, and classification naming is Enterococcus faecium,
It is CGMCC1.15321 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The 16S rDNA gene order of the enterococcus faecium is as shown in SEQ ID No.1, atpA gene order such as SEQ ID
Shown in No.2.
The enterococcus faecium is isolated from through 5kGy gamma-ray irradiation treated anaerobically digested sludge.
The biological characteristics of the enterococcus faecium are as follows: cell is oval under microscope or club-shaped, no gemma, no whip
Hair；Bacterial strain Gram's staining is positive, amphimicrobian；Bacterial strain cultivates 20-48h under 25-40 DEG C of anaerobic condition, can form surface
Milky bacterium colony that is smooth, having protrusion, neat in edge, colony diameter 1-1.5mm.
Application of the enterococcus faecium in hydrogen production through anaerobic fermentation as described above.
The enterococcus faecium under anaerobic fermentation and hydrogen production the step of are as follows:
Enterococcus faecium is seeded in enriched medium, Anaerobic culturel is to bacterium under the conditions of 25-40 DEG C, 80-120r/min
The logarithmic phase of strain growth；With the inoculation of 5-30% than accessing in the fermentation medium of initial pH=5-10, at 25-40 DEG C of temperature,
Anaerobic fermentation 20-48h under the conditions of 80-120r/min；
The component of the enriched medium are as follows: debirs COD=50g/L, peptone 10g/L, yeast powder 0.5g/L, battalion
Nutrient solution 10mL/100mL；
The component of the fermentation medium are as follows: debirs COD=5-20g/L, nutrient solution 10mL/100mL.
Enriched medium and fermentation medium sterilize 30min through high pressure water-bath under the conditions of 115 DEG C.
Fermentation and hydrogen production under the anaerobic condition does not need light source.
In the debirs, carbohydrate concentration COD=5-20g/L；The component of the nutrient solution are as follows: NaHCO3
40g/L, NH4Cl 5g/L, NaH2PO4·2H2O 5g/L, K2HPO4·3H2O 5g/L, FeSO4·7H2O 0.25g/L,
MgCl2·6H2O 0.085g/L, NiCl2·6H2O 0.004g/L。
In the range of the initial pH=5-10 of fermentation medium, as initial pH=7, fermentation and hydrogen production amount is maximum.
5-30% inoculation than in range, when inoculation is than being 10%, fermentation and hydrogen production amount is maximum.
Within the temperature range of 25-40 DEG C, when the temperature of anaerobic fermentation is 35 DEG C, fermentation and hydrogen production amount is maximum.
The invention has the benefit that
Filter out one plant of new enterococcus faecium, the enterococcus faecium have growth rapidly, it is hydrogen generation efficiency height, drug resistance, resistance to
The features such as heat, resistance to acid and alkali；Requirement of the fermentation and hydrogen production system to environment can be reduced, the economic benefit of whole system is promoted；Together
When can using debirs as substrate be made clean energy resource hydrogen, can achieve the double benefit of Waste disposal and production of energy.
Biomaterial preservation explanation
Classification naming: enterococcus faecium (Enterococcus faecium)；Strain number: INET2
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on June 3rd, 2015
Collection is registered on the books number: CGMCC1.15321.
Figure of description
Fig. 1 is the biological evolution tree graph of enterococcus faecium (Enterococcus faecium) INET2.
Fig. 2 is the growth curve chart of enterococcus faecium (Enterococcus faecium) INET2.
Fig. 3 is the fermentation and hydrogen production device figure of enterococcus faecium (Enterococcus faecium) INET2.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.
Embodiment 1: the separation of enterococcus faecium (Enterococcus faecium) INET2 and bacterial strain are identified:
It is sampled from the sludge primary digestion pond of Beijing's municipal sewage plant, the anaerobically digested sludge of acquirement is in room
Under temperature state, through radionuclide60The gamma-ray irradiation of Co transmitting, dosage 5kGy.Sludge after irradiation is in enriched medium
Middle anaerobism enrichment culture 20-48h, the fermentation and hydrogen production 20-48h in the culture medium using glucose as sole carbon source, then bacterium solution is dilute
It releases and is coated on solid plate culture medium.Pass through 20-48h Anaerobic culturel, picking isolated single bacterium under the conditions of 25-40 DEG C
It falls, is inoculated in fermentation medium, the fermented and cultured 20-48h at 25-40 DEG C, using gas-chromatography (112A, Shanghai, heat
Lead detector, packed column TDX-01 3000*3mm, 160 DEG C of column temperature, 110 DEG C of detector, 180 DEG C of sample injector) it detects in fermentation flask
The gas component of generation, by the screening process of a large amount of bacterial strains, isolated one plant of hydrogen production potential is strong and low to oxygen sensitive degree
Bacterial strain INET2.
To the bacterial strain that separation screening obtains, China Committee for Culture Collection of Microorganisms's common micro-organisms center is entrusted
(CGMCC) strain idenfication is carried out, according to the cellular morphology of strain, physiological and biochemical property, 16S rRNA gene order and atpA base
Because of the test datas comprehensive analysis such as sequence, identify that bacterial strain belongs to Firmicutes (Firmicutes), enterococcus spp
(Enterococcus) enterococcus faecium (Enterococcus faecium), is named as INET2.
The biological characteristics of bacterial strain INET2 are as follows: cell is oval under microscope or club-shaped, no gemma, atrichia；
Bacterial strain Gram's staining is positive, amphimicrobian；Bacterial strain cultivates 20-48h under 25-40 DEG C of anaerobic condition, can form surface light
Milky bacterium colony that is sliding, having protrusion, neat in edge, colony diameter 1-1.5mm.
The physio-biochemical characteristics qualification result of bacterial strain INET2 is as shown in table 1, and 16S rDNA sequence length is 1437kp,
Full length sequence is as shown in SEQ ID NO.1, and atpA sequence length is 861kp, and full length sequence is as shown in SEQ ID NO.2.
According to obtained 16S rRNA sequence, the matching analysis is carried out in the GenBank of the website ezbiocloud, draws it
Biological evolution tree (such as Fig. 1), obtain position and affiliation of the bacterial strain during biological evolution.It will be seen from figure 1 that
Enterococcus faecium INET2 (CGMCC 1.15321) and enterobacter asburiae have closer affiliation, and enterobacter asburiae is then a kind of
The generally acknowledged strain with high hydrogen production potential.
1 physio-biochemical characteristics qualification result of table
| Index | As a result | Index | As a result |
| Gram's staining | It is positive | Carbohydrate produces sour (Continued) | |
| Cell shape | It is spherical | L-arabinose | + |
| Form gemma | - | L- sorbose | - |
| Catalase | - | L- rhamnose | - |
| Oxidizing ferment | - | Lactose | + |
| It is grown in air | + | Sucrose | + |
| 50 DEG C of growths | - | Maltose | + |
| 45 DEG C of growths | + | Trehalose | + |
| 15 DEG C of growths | + | Melibiose | + |
| 6.5%NaCl growth | + | Cellobiose | + |
| Carbohydrate produces acid | Melezitose | - | |
| D-Glucose | + | Gossypose | + |
| D-Fructose | + | Sorbierite | - |
| D-MANNOSE | + | Mannitol | + |
| D-ribose | + | Sodium gluconate | - |
| D- xylose | - | Aesculin | + |
| L- xylose | - | Salicin | + |
| D- galactolipin | + | Amarogentin | + |
| D-arabinose | - | Starch | - |
Embodiment 2: the drafting of enterococcus faecium (Enterococcus faecium) INET2 growth curve
Enterococcus faecium (Enterococcus faecium) INET2 is inoculated into enriched medium, at 36 DEG C, 100r/
Constant-temperature shaking culture under the conditions of min sampled every 2 hours, measures its absorbance at 600nm, draw the growth of the bacterial strain
Curve, as shown in Figure 2.
Embodiment 3: the fermentation and hydrogen production situation of enterococcus faecium (Enterococcus faecium) INET2 at different temperatures
The bacterial strain in logarithmic growth phase is taken, is inoculated in fermentation medium with 10% inoculation ratio, wherein glucose is dense
Degree is 10 g/L, and the initial pH of fermentation medium is 7.0.The constant temperature under the conditions of condition of different temperatures (25-40 DEG C), 100r/min
Oscillating reactions, reaction unit are as shown in Figure 3.The gas that reaction process generates first passes through sodium hydroxide drexel bottle, absorbs in gas
Carbon dioxide, then collect residual gas with draining water gathering of gas law, the content of hydrogen uses gas Chromatographic Determination in collected gas.
Fermentation and hydrogen production situation such as table 2 after 48h fermentation reaction, under condition of different temperatures.
The fermentation and hydrogen production situation of 2 enterococcus faecium of table (Enterococcus faecium) INET2 at different temperatures
Embodiment 4: fermentation and hydrogen production feelings of enterococcus faecium (Enterococcus faecium) INET2 at different initial pH
Condition
The bacterial strain in logarithmic growth phase is taken, is inoculated in fermentation medium with 10% inoculation ratio, wherein glucose is dense
Degree is 10g/L, at 35 DEG C, constant-temperature shaking culture under the conditions of 100r/min.It is adjusted just with 1mol/L hydrochloric acid and sodium hydroxide solution
Beginning pH value (5.0-10.0), the fermentation and hydrogen production situation of bacterial strain under the conditions of more different initial pH, what is obtained the results are shown in Table 3.
Fermentation and hydrogen production situation of 3 enterococcus faecium of table (Enterococcus faecium) INET2 at different initial pH
Embodiment 5: fermentation of enterococcus faecium (Enterococcus faecium) INET2 under different concentration of glucose produces
Hydrogen situation
The bacterial strain in logarithmic growth phase is taken, is inoculated in fermentation medium with 10% inoculation ratio, fermentation medium
Initial pH is 7.0.At 35 DEG C, constant-temperature shaking culture under the conditions of 100r/min.Compare different initial glucose concentrations (5-20g/L)
Under the conditions of bacterial strain fermentation and hydrogen production situation, what is obtained the results are shown in Table 4.
Fermentation and hydrogen production feelings of 4 enterococcus faecium of table (Enterococcus faecium) INET2 under different concentration of glucose
Condition
Embodiment 6: fermentation and hydrogen production feelings of enterococcus faecium (Enterococcus faecium) INET2 in different vaccination than under
Condition
The bacterial strain in logarithmic growth phase is taken, is inoculated in fermentation medium with different inoculations than (5-30%), wherein
Concentration of glucose is 10g/L, and the initial pH of fermentation medium is 7.0.At 35 DEG C, constant-temperature shaking culture under the conditions of 100r/min.
Compare the fermentation and hydrogen production situation of bacterial strain under the conditions of different initial strains inoculations are compared, what is obtained the results are shown in Table 5.
Fermentation and hydrogen production situation of 5 enterococcus faecium of table (Enterococcus faecium) INET2 in different vaccination than under
Claims (7)
1. a kind of manure enterococcin strain, enterococcus faecium (Enterococcus faecium) bacterial strain is INET2, in China
The deposit number of General Microbiological Culture preservation administrative center is CGMCC No.1.15321.
2. application of the manure enterococcin strain described in claim 1 in hydrogen production through anaerobic fermentation.
3. application according to claim 2, which is characterized in that manure enterococcin strain fermentation and hydrogen production under anaerobic
The step of are as follows:
Manure enterococcin strain is seeded in enriched medium, Anaerobic culturel is to bacterium under the conditions of 25-40 DEG C, 80-120r/min
The logarithmic phase of strain growth；With the inoculation of 5-30% than accessing in the fermentation medium of initial pH=5-10, at 25-40 DEG C of temperature,
Anaerobic fermentation 20-48h under the conditions of 80-120r/min；
The component of the enriched medium are as follows: debirs COD=50g/L, peptone 10g/L, yeast powder 0.5g/L, nutrient solution
10mL/100mL；
The component of the fermentation medium are as follows: debirs COD=5-20g/L, nutrient solution 10mL/100mL；
The component of the nutrient solution are as follows: NaHCO340g/L, NH4Cl 5g/L, NaH2PO4·2H2O 5g/L, K2HPO4·3H2O
5g/L, FeSO4·7H2O 0.25g/L, MgCl2·6H2O 0.085g/L, NiCl2·6H2O 0.004g/L。
4. application according to claim 3, which is characterized in that the fermentation and hydrogen production under the anaerobic condition does not need light source.
5. application according to claim 3, which is characterized in that the initial pH=7 of the fermentation medium.
6. application according to claim 3, which is characterized in that the inoculation is than being 10%.
7. application according to claim 3, which is characterized in that the temperature of the anaerobic fermentation is 35 DEG C.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981117A (en) * | 2013-12-24 | 2014-08-13 | 北京伟嘉人生物技术有限公司 | High stress resistant enterococcus faecium and culture method and application thereof |
| CN104263705A (en) * | 2014-09-16 | 2015-01-07 | 清华大学 | Method for selectively producing lignin-degrading enzymes by using phanerochaete chrysosporium |
| CN104560820A (en) * | 2014-12-30 | 2015-04-29 | 杭州师范大学 | Enterococcus faecium KQ2.6 and application thereof |
| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
-
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- 2015-12-11 CN CN201510920684.1A patent/CN105754889B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981117A (en) * | 2013-12-24 | 2014-08-13 | 北京伟嘉人生物技术有限公司 | High stress resistant enterococcus faecium and culture method and application thereof |
| CN104263705A (en) * | 2014-09-16 | 2015-01-07 | 清华大学 | Method for selectively producing lignin-degrading enzymes by using phanerochaete chrysosporium |
| CN104560820A (en) * | 2014-12-30 | 2015-04-29 | 杭州师范大学 | Enterococcus faecium KQ2.6 and application thereof |
| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
Non-Patent Citations (3)
| Title |
|---|
| Enriching hydrogen-producing bacteria from digested sludge by different pretreatment methods;Yanan Yin等;《International Journal of Hydrogen Energy》;20140220;第39卷(第25期);第13551页右栏第1-2段 * |
| 李宇亮等.高效产氢菌株Enterococcus sp.LG1的分离及产氢特性.《微生物学通报》.2008,第35卷(第9期),摘要. * |
| 高效产氢菌株Enterococcus sp.LG1的分离及产氢特性;李宇亮等;《微生物学通报》;20080920;第35卷(第9期);摘要 * |
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