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Multifunctional clostridium bifermentans and application thereof

A double-enzyme clostridium, multi-functional technology, applied in the field of microorganisms, to achieve the effect of fast growth, strong application potential, and enhanced yield of target products

Active Publication Date: 2017-09-29
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently discovered electroactive microorganisms are mainly concentrated in Gram-negative Geobacter and Shewanella, while the electrochemically active Gram-positive Clostridium Clostridium is rarely reported.

Method used

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  • Multifunctional clostridium bifermentans and application thereof
  • Multifunctional clostridium bifermentans and application thereof
  • Multifunctional clostridium bifermentans and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Isolation and identification of Clostridium bifermentans EZ-1:

[0035] 1) Take the enriched Yellow River Delta soil supernatant, press 10-10 6 1-fold serial dilution, the inoculation loop was dipped to dilute 10 6 1 times the soil supernatant, marked on a PYG petri dish containing 2% agarose, and cultured in an anaerobic incubator at 30°C for 24h to 48h to obtain a single colony, which was round and white as figure 1 shown.

[0036] 2) Select a single colony and transfer it into 20 μL of sterile water, take 2 μL of the bacterial suspension for PCR amplification, and insert the remaining 18 μL into 10 ml of PYG liquid medium, culture at 30°C for 24-48 hours in the dark, and observe the bacterial morphology under an inverted microscope like figure 2 shown.

[0037] 3) PCR system: 2 μL buffer buffer; 2 μL dNTP substrate; 0.5 μL Taq enzyme; 0.5 μL Ba907r reverse primer; 0.5 μL Ba27F forward primer; 2 μL single colony suspension template; 2.5 μL sterile water.

[003...

Embodiment 2

[0042] Anaerobic fermentation and product identification of Clostridium bifermentans EZ-1:

[0043] 1) Medium PYG: peptone: 0.2-5g / L, tryptone: 0.2-5g / L, yeast extract: 0.2-10g / L, glucose: 5-10g / L, CaCl 2 : 0.008g / L, MgSO 4 ·7H 2 O: 0.016g / L, K 2 HPO 4 : 0.04g / L, KH 2 SO 4 : 0.04g / L, NaHCO 3 : 0.04g / L, NaCl: 5g / L, L-cysteine: 0.25g / L; PH 6.3 (±0.2)

[0044] 2) Divide the above PYG medium into 25ml serum bottles, 10ml in each bottle, and add silica gel stoppers. Deoxygenation: evacuate for 6 minutes, flush with nitrogen for 30 seconds; pump for 3 minutes, flush with nitrogen for 30 seconds; pump for 1 minute, flush with nitrogen for 30 seconds. The aluminum cover is sealed, and sterilized by high-pressure steam at 121°C for 20 minutes.

[0045] 3) Inoculate Clostridium bifermentans EZ-1 into the deoxygenating syringe, the inoculum size is 1%, and the growth situation is measured, and the results are as follows: image 3 shown. Take 200 μ L of top gas for gas chromato...

Embodiment 3

[0048] Detection of dissimilatory iron reduction ability of Clostridium bifermentans EZ-1:

[0049] 1) Add respectively to the PYG medium after autoclaving: magnetite (Fe 3 o 4 ) final concentration is 2.5-10mm / L; final concentration of ferrihydrite (FeOOH) is 5.0-10mm / L; final concentration of ferric citrate is 10-40mm / L.

[0050] 2) Then inoculate Clostridium dizymogenum cultured to the logarithmic phase in the PYG liquid medium, the inoculum amount is 1%, 30° C., and cultivate in the dark for 48 hours.

[0051] 3) Use the phenanthrozine method to detect the changes in the concentration of ferrous iron in the culture medium before and after cultivation as follows: Image 6 shown.

[0052] It can be seen from the above figures that the bienzyme Clostridium has strong iron reduction ability, not only can reduce soluble and amorphous iron, but also can reduce crystalline iron in nano-magnetite. It can be seen that the Gram-positive bacterium Clostridium bienzyme of the bac...

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Abstract

The invention belongs to the technical field of microorganisms, and specifically relates to multifunctional clostridium bifermentans and application thereof in producing hydrogen through fermentation and increasing the yield of acetic acid. A bacterial strain is clostridium bifermentans EZ-1, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO. 13913. The multifunctional clostridium bifermentans is capable of generating clean fuel hydrogen and metabolites such as acetic acid and butyric acid by utilizing glucose. Due to addition of nano magnetite and ferrihydrite in appropriate concentrations, a metabolism path of the multifunctional clostridium bifermentans can be regulated, so that the yield of the acetic acid can be increased. By microbial fuel cell detection, the situation that the bacterial strain has power generation ability is discovered, and the maximum current output density can be up to 6.3 mA / m<2>. The bacterial strain is first multifunctional novel clostridium bifermentans which is reported at present, can be applied to biological products of hydrogen, acetic acid and butyric acid generated through biomass fermentation, has power generation potential and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a multifunctional novel double-enzyme clostridium and its application in fermenting hydrogen production and increasing acetic acid production. Background technique [0002] Clostridium bienzyme is a strictly anaerobic fermentative hydrogen-producing bacterium that can produce hydrogen and volatile short-chain fatty acids from fermentation substrates such as glucose, and its Gram stain is positive. [0003] With the increasingly prominent climate change and air pollution problems caused by the use of fossil fuels, biohydrogen has great application prospects because of its clean and renewable characteristics and high combustion heat (122kJ / g). Biohydrogen is mainly produced by hydrogen-producing microorganisms through dark fermentation. Such microorganisms mainly include Clostridium, Enterobacter, and Bacillus, among which Clostridium is easy to cultivate and has high ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P3/00C12P7/40C12P7/54H01M8/16C12R1/145
CPCC12P3/00C12P7/40C12P7/54C12N1/205C12R2001/145H01M8/16Y02E60/50
Inventor 刘芳华张月超王欧美杨翠云肖雷雷
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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