Multifunctional Dizyme Clostridium and Its Application

A double-enzyme Clostridium, multifunctional technology, applied in the field of microorganisms, to achieve the effects of easy cultivation, fast growth and high hydrogen production rate

Active Publication Date: 2020-05-05
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 Dizyme Clostridium and Its Application
  • Multifunctional Dizyme Clostridium and Its Application
  • Multifunctional Dizyme Clostridium and Its Application

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 Such as 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.

[...

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 microbes, and in particular relates to a multifunctional novel double-enzyme clostridium and its application in fermenting hydrogen production and increasing acetic acid production. The bacterial strain is Clostridium bifermentans EZ-1, and the preservation number is CGMCC NO.13913. The bacteria obtained in the invention can utilize glucose to produce clean fuel hydrogen and metabolites such as acetic acid and butyric acid. Adding appropriate concentrations of nano-magnetite and ferrihydrite can regulate the metabolic pathway of the bacteria to increase the production of acetic acid. Through the detection of microbial fuel cells, it was found that the strain has the ability to produce electricity, and the maximum current output density can reach 6.3mA / m 2 . This strain is the first reported case of a new multi-functional double-enzyme Clostridium, which can be applied to biomass fermentation to produce biological products such as hydrogen, acetic acid and butyric acid, and has the potential to produce electricity, so it has broad application prospects.

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 Patents(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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