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Method for collecting cellulose degradation bacterium strain

A technology of cellulose degrading bacteria and cellulase, applied in the field of environmental microorganisms, can solve the problems of unfavorable bacterial proliferation, high viscosity of CMC liquid medium, unfavorable collection of bacteria, etc.

Active Publication Date: 2020-10-20
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] 1. When using a solid culture plate to collect samples, repeated washing and centrifugation with sterile water are required, so that it takes about 5 minutes to collect a sample, which makes it difficult for the results of enzyme activity characterization to reflect the enzyme activity under actual culture conditions;
[0005] 2. When using liquid CMC medium to collect samples, due to the high viscosity of CMC liquid medium, it is not conducive to the proliferation of bacteria, and it is also not conducive to collecting bacteria by centrifugation

Method used

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  • Method for collecting cellulose degradation bacterium strain
  • Method for collecting cellulose degradation bacterium strain
  • Method for collecting cellulose degradation bacterium strain

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0029] Embodiment 1: Characterize the cellulose degrading ability of bacterium described in the present invention

[0030] 1. The composition of the CMC medium used in the embodiment is: CMC-Na 15 g, NH 4 NO 3 1 g, yeast extract 1 g, MgSO 4 ·7H 2 O 0.5 g, KH 2 PO 4 1 g, 15 g agar, distilled water to 1 L, 121 ℃ autoclave for 20 min;

[0031] 2. The concentration of Congo red dye solution used in this example is 1 mg / ml, and the concentration of NaCl solution is 1mol / L;

[0032] 3. The cellulose-degrading bacteria used in this example are Bacillus subtilis (preservation number CGMCC No. 18089) and alpine bacillus (preservation number CGMCC No. 17046).

[0033] 4. Expand the culture of the bacteria in LB liquid medium, shake at 37 °C and 150 rpm for 12 h.

[0034] 5. Collect the bacterial solution cultivated overnight into a 10 ml sterilized centrifuge tube, centrifuge at 6000 rpm for 5 min at room temperature, collect the bacterial pellet, and discard the supernatant. ...

Embodiment 2

[0041] Embodiment 2: the cellulase activity of characterizing bacterium described in the present invention

[0042] On the CMC solid plate, use the method in Example 1 to cultivate cellulose-degrading bacteria for 72 h, quickly clamp out the filter paper with sterile tweezers, put it into a sterilized centrifuge tube, and use the DNS method to determine the bacterial fiber Sulfase activity.

[0043] 1, the CMC culture medium that uses in the embodiment is composed with embodiment 1;

[0044] 2. The cellulose-degrading bacteria used in this example are the same as in Example 1;

[0045] 3. In this example, use an ultraviolet spectrophotometer (UV) to measure the absorbance of the treated sample;

[0046] 3. The absorbance and cellulase activity measured in the present embodiment are as follows:

[0047] Table 1 Enzyme activity detection results

[0048]

[0049] The above proves that the bacteria collected by the method of the present invention can carry out quantitative...

Embodiment 3

[0050] Embodiment 3: the sample that the method of the present invention collects is used for extracting DNA

[0051] On the CMC solid plate, use the method of Example 1 to cultivate the cellulose-degrading bacteria for 72 h, quickly clamp the filter paper out with sterile tweezers, put it into a sterilized centrifuge tube, and use phenol-chloroform to extract the bacteria on the filter paper. bacterial DNA.

[0052] 1, the CMC culture medium that uses in the embodiment forms with embodiment 1;

[0053] 2. In this example, Nanodrop 2000 was used to measure the DNA content;

[0054] 3. The cellulose-degrading bacteria used in this example are the same as in Example 1;

[0055] 4. Using 1% agarose gel electrophoresis in the examples, clear and bright DNA bands can be obtained ( figure 2 );

[0056]5. Compared with the traditional method of collecting bacteria (coating method), the method of using filter paper can stably extract DNA and can be used for subsequent PCR experim...

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Abstract

The invention provides a method for collecting a cellulose degradation bacterium strain, and belongs to the technical field of environment microorganisms. The method comprises the specific steps of firstly, utilizing an LB liquid nutrient medium, and performing extended culture on cellulose degradation bacteria; performing centrifugation to remove a culture medium, and uniformly spraying and sucking the strain as inoculating liquid; dipping in the bacterial liquid with bacteria-free filter paper, tiling the bacteria-free filter paper on a CMC solid culture plate, and performing culture at 37 DEG C for 72h; and collecting the filter paper to obtain samples required for detecting the activity of cellulase of the cellulose degradation bacteria. The method is simple to operate, stable in results, time-saving and reliable, and has broad application value in the respect of representing the activity of the cellulase of the cellulose degradation bacteria.

Description

technical field [0001] The invention relates to the field of environmental microorganisms, in particular to a method for rapidly collecting cellulose-degrading bacteria. Background technique [0002] Cellulose-degrading bacteria refer to bacteria that can degrade cellulose. Through the action of cellulase produced in the bacteria, the difficult-to-degrade cellulose is decomposed into cellobiose and then decomposed into glucose that is easy to decompose and utilize. Cellulose-degrading bacteria are widely distributed in the intestines of humus and herbivorous animals, and can be divided into aerobic degrading bacteria and anaerobic degrading bacteria. There are a lot of cellulose resources on the earth, more than half of which are not utilized, resulting in waste of resources and environmental pollution. The degradation of cellulose in nature is mainly through biodegradation, so the screening of highly efficient cellulose-degrading bacteria plays an important role in the tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12Q1/34C12R1/125C12R1/07
CPCC12N1/20C12N9/2437C12Q1/34Y02W30/40
Inventor 石振华张琪周顺桂
Owner FUJIAN AGRI & FORESTRY UNIV
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