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A method and application for real-time monitoring of biogas fermentation system

A technology of real-time monitoring and biogas fermentation, applied in biochemical equipment and methods, measurement/inspection of microorganisms, introduction of foreign genetic material using carriers, etc., can solve problems such as high cost, long detection cycle, and inability to accurately reflect cell biomass , to achieve the effect of accurate monitoring and optimization of gas production

Inactive Publication Date: 2020-02-14
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have the disadvantages of long detection cycle, high cost, and inaccurate reflection of cell biomass.

Method used

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  • A method and application for real-time monitoring of biogas fermentation system
  • A method and application for real-time monitoring of biogas fermentation system
  • A method and application for real-time monitoring of biogas fermentation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Correlation between Clostridium cellulolyticum cell growth density and fluorescence density

[0034] Clostridium cellulolyticum was purchased from German Culture Collection (DSM5812). The gfp gene was amplified using the plasmid pMUTIN-gfp as a template.

[0035] The upstream primer is F: 5'-CG GGATCC AAGAAGATATACATATGGCT-3' (SEQ ID NO. 1);

[0036] The downstream primer is R: 5'-CG GGATCC CTCGAATTCATTATTTGTAG-3' (SEQ ID NO.2), the underline is the restriction site of BamH I.

[0037] The amplification system was 25 μL, and the reaction program was as follows: template pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 90 seconds. A total of 30 cycles, with a final extension at 72°C for 10 min. PCR amplified products were separated by 1% agarose gel electrophoresis and purified using a purification kit. The PCR product and the pET15b vector were respectively digested...

Embodiment 2

[0040] Example 2: Monitoring the activity of GFP-labeled Clostridium cellulolyticum (DAM5812-GFP) in a leaky reactor

[0041] Corn stalks were crushed into 1×1cm block material, mixed with activated sludge according to the VS ratio of 2:1, then an appropriate amount of water was added to adjust the total TS content to 8%, and NH4Cl was added to supplement the nitrogen source, and NaHCO 3 Adjust the alkalinity to 3000mg CaCO 3 / L. Use 2M NaOH solution to absorb H 2 S, CO 2 Such as acid gas, the gas production is measured by the lye discharge method, which is the methane production. Cultivate DSM5812-GPF under anaerobic conditions until OD 600 When the growth reached 0.45, the cells were collected by centrifugation, and the cells were resuspended with a small amount of medium. Under the protection of N2, the bacterial solution was added to the seepage anaerobic reactor. Bacteria are evenly distributed by the seepage fluid circulation method. At 0, 2, 5, 10, and 15 days of...

Embodiment 3

[0043] Example 3 Effect of raw material pretreatment on hydrolyzing microorganisms in biogas fermentation flora

[0044] The corn stalks were crushed into a 1×1 cm block, and water was added to adjust the TS to 10%. Add 6% NaOH (w / w), at 30°C, 100 rpm, for 3d. After the end, the solid-liquid separation is carried out by pressing to obtain the alkali-treated straw. Alkali-treated straw and untreated straw were used as fermentation substrates, mixed with activated anaerobic sludge at a VS ratio of 2:1, and fermented in a seepage reactor. Fermentation conditions, strain addition and sampling determination are the same as those described in Example 2.

[0045] The results showed that the concentration of DSM5812-GFP detected in the initial alkaline treatment reactor was lower, indicating that the straw surface was more susceptible to microbial adsorption after pretreatment. As the fermentation progressed, alkali treatment of straw destroyed the physical barrier effect of lignin...

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Abstract

The invention discloses a method for monitoring a biogas fermentation system in real time and an application and belongs to the technical field of biogas fermentation. According to the method, a GFP (green fluorescent protein) gene is amplified and connected and transferred into a host microbe, positive clones are selected, a GFP gene marked engineering bacterium is constructed, and then the GFP gene marked engineering bacterium is applied to the biogas fermentation system to monitor the fermentation system in real time. With the adoption of the method, the problem that space and time distribution of the microbial biomass and metabolic activity in the anaerobic fermentation is difficult to monitor in real time is solved, the GFP gene marked engineering bacterium is applied to monitoring of the biogas fermentation system, the fermentation state and target microbes in the fermentation system can be monitored rapidly and accurately, the number of microbes is tracked quantitatively, unstable factors during fermentation are discovered timely, and biogas production is further optimized.

Description

technical field [0001] The invention relates to a method and application for real-time monitoring of a biogas fermentation system, belonging to the technical field of biogas fermentation. Background technique [0002] As the fossil energy crisis continues to intensify and environmental issues become increasingly prominent, renewable and clean energy is getting more and more attention. However, biogas fermentation has outstanding advantages due to its high energy output / input ratio (28:1), a wide range of available substrates, and clean products. At present, tens of thousands of large and medium-sized biogas projects are in operation in China, but there are common problems such as low treatment efficiency and poor operation stability. Biogas engineering is a biochemical process that is crucial to the monitoring and control of microorganisms. Due to the interaction of multiple factors such as microbial adaptability and fermentation conditions, it is usually difficult to find...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12Q1/04
CPCC12N15/63C12Q1/04
Inventor 李十中韩娅新张成明姜立陈雪兰
Owner TSINGHUA UNIV
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