CN116103157A - A screening method for trichoderma strains degrading bagasse cellulose - Google Patents

Abstract

The invention discloses a Trichoderma strain capable of degrading bagasse cellulose, which is named Trichoderma A-8 (Trichoderma sp.A-8), and is obtained by separating a strain with a larger transparent ring after static culture of mildewed bagasse, and then screening out the strain with high filter paper weight loss rate and high enzyme activity from the strain, wherein the obtained strain is Trichoderma strain A-8. The obtained trichoderma strain A-8 has stable enzyme producing capability and high enzyme producing capability. The trichoderma strain A-8 screened by the method has simple process and easy realization, and is suitable for degrading bagasse cellulose.

Description

A screening method for trichoderma strains degrading bagasse cellulose

technical field

The invention belongs to the technical field of microbes, and in particular relates to a screening method for Trichoderma strains that degrade bagasse cellulose.

Background technique

Bagasse is the main by-product of the sugar cane sugar industry process. It is the solid fiber residue of sugarcane stems left after sugarcane is mechanically crushed, multi-roll pressed and solid-liquid (cane juice) separated. It is a renewable, An abundant resource suitable for the production of bio-based products such as biofuels and chemicals, an ideal alternative to traditional petroleum-based fuels and the backbone of the biorefinery concept. Except for a very small part of bagasse resources used for biorefining and pulping and papermaking, they are mainly used for burning in sugarcane sugar factories and bioethanol factories to generate heat and electricity. The added value of use is low and it is easy to cause secondary pollution. Therefore, it is necessary to choose an ecological approach to deal with it. A mutant strain with high cellulase production and stable inheritance is obtained, which is used to degrade bagasse biomass, thereby improving the conversion rate of bagasse cellulose, and making bagasse achieve high value and resource utilization.

The high-value and resource utilization of cellulosic biomass has become a research hotspot, and the bagasse cellulosic material is energy-saving and environmentally friendly. Among the various methods for degrading cellulosic biomass, biological methods are more effective. Degradation and transformation utilize cellulase, a secondary metabolite of microorganisms, but it has not been widely used in industrial production. The main reason is that in practical applications, there are strains with weak enzyme production ability and unstable quality. Therefore, it is urgent to find fungi with high-efficiency cellulase production potential. For decades, researchers have screened various fungal cultures for the enzyme, but most fungal cultures have yet to be discovered. Screening wild strains with strong enzyme production ability and high enzyme activity can bring higher commercial profits to enterprises, and is of great significance to the effective utilization of bagasse cellulose resources.

However, there are still problems such as low resource utilization rate of bagasse, difficulty in degrading cellulose, and high cost, mainly due to high enzyme cost and low sugar production efficiency. In order to further expand the utilization of bagasse, it is a feasible way to obtain enzyme-producing strains that can efficiently degrade bagasse fibers.

Contents of the invention

The technical problem to be solved in the present invention is to provide a strain of cellulase-producing fungus Trichoderma A-8, which can effectively degrade bagasse cellulose, release oligosaccharides or monosaccharides, and has high enzyme production capacity and low enzyme cost.

In order to achieve the above object, the present invention adopts the following technical means: a strain of cellulase producing bacteria Trichoderma A-8, preserved in China General Microorganism Culture Collection Management Center CGMCC No.20746, the address is: Chaoyang District, Beijing No. 3, No. 1 Courtyard, Beichen West Road, the preservation time is October 26, 2020.

A screening method for cellulase producing bacteria Trichoderma A-8, comprising the following steps:

(1) Get the moldy bagasse sample and place it in the sample bottle, and culture it statically in the culture medium;

(2) Gradient dilution of the obtained bacterial solution was carried out, coated on a CMC-Na solid plate, placed in a constant temperature incubator for cultivation, and then cultured by streaking on a slant plane, and a single colony was separated and purified according to the colony shape and color;

(3) The purified bacterial strain obtained by separation is taken and inserted into the Congo red culture medium with a puncher to cultivate, and the bacterial strain with a larger transparent circle is isolated;

(4) Screen strains with high filter paper weight loss rate and high enzyme activity from the strains isolated in step (3), then place the screened strains in a constant temperature shaking incubator for cultivation, and then insert PDA medium for repurification get.

The culture medium of the static culture is a liquid enrichment medium with CMC-Na as a carbon source.

The cultivation in the constant temperature incubator was carried out at 30° C. for 5 days.

The Congo red medium is prepared according to the following ratio: (NH ₄ ) ₂ SO ₄ 2.0g, MgSO ₄ ·7H ₂ O 0.5g, K ₂ HPO ₄ 1.0g, NaCl 0.5g, microcrystalline cellulose 2.0g , Congo red 0.4g, agar 20g, add ultrapure water to 1000mL; after preparation, sterilize at 121°C for 30min.

The conditions for culturing in the constant temperature shaking incubator are 30° C., 200 r/min.

The PDA medium is to weigh 200g of peeled potatoes, 20g of glucose, 20g of agar, and the pH is natural. The potatoes are cut into small cubes, boiled in water for about 30min, then filtered with four layers of gauze, and glucose and agar are added to the filtrate. powder, heat, stir with a glass rod until the glucose and agar powder are completely dissolved, and finally add ultrapure water to 1000mL, and sterilize at 121°C for 30min.

The beneficial effects of the present invention are:

Provides a cellulase-producing strain Trichoderma A-8, and provides a screening method for the strain, the strain can effectively degrade bagasse cellulose, release oligosaccharides or monosaccharides, and have high enzyme-producing ability, Enzymes are low cost.

Description of drawings

FIG. 1 is a phylogenetic tree based on ITS sequences in Example 4.

Detailed ways

The present invention will be described below in conjunction with the accompanying drawings and embodiments.

Example 1:

This embodiment is an embodiment of the screening method of a cellulase-producing fungus Trichoderma A-8 according to the present invention, comprising the following steps:

(1) Take the moldy bagasse sample and place it in a sample bottle, and culture it statically in the medium: use CMC-Na as the carbon source, and use the liquid enrichment medium to obtain the cellulolytic bacteria. Place it on a vortex shaker and oscillate at the maximum frequency for 5 minutes to elute the microorganisms on the sample, take out 2mL of the bacterial liquid with a pipette gun, inoculate it in a fresh 100mL CMC-Na liquid medium, place it in a 250mL flask, and put it in a 250mL flask for 30 ℃, cultivated for 7 days to make it enrich.

(2) The obtained bacterial solution was serially diluted, and 100 μL of 10 ^-4 , 10 ^-5 , and 10 ^-6 dilutions were drawn and spread on the CMC-Na solid plate, and placed in a constant temperature incubator at 30°C for 5 days. , and then carry out slant streak culture, and isolate and purify a single colony according to the colony shape and color.

(3) Use a hole puncher to take the isolated purified strains and insert them into the Congo red medium, place them in a constant temperature incubator at 30°C and cultivate them for 5 days, measure the size of the transparent circle, and isolate the one with the largest transparent circle. strain; Congo red medium was prepared according to the following proportions: (NH ₄ ) ₂ SO ₄ 2.0g, MgSO ₄ ·7H ₂ O 0.5g, K ₂ HPO ₄ 1.0g, NaCl 0.5g, microcrystalline cellulose 2.0g, Congo red 0.4g, agar 20g, add ultra-pure water to 1000mL; after preparation, sterilize with damp heat at 121°C for 30min.

(4) The filter paper disintegration experiment was carried out using the strains screened out to verify its degradation ability. Each test tube is divided into 7mL filter paper medium, put 50mg (1cm × 6cm) filter paper (weighed after drying) as the enzymolysis medium, inoculate the strains of different forms on the filter paper, and incubate at 30°C. cultivated in. The test tube showing that the filter paper was decomposed was diluted, and streak culture was carried out in CMC-Na medium. After culturing at 30°C for 72 hours, the grown single colony was inoculated on the filter paper medium and cultured for 10 days. The filter paper was collected by filtration, washed with ultrapure water, dried at 80°C and weighed. It is also necessary to further measure the cellulase activity (CMC enzyme activity) of the strains to screen out the strains with large transparent circles, high filter paper weight loss rate and high enzyme activity. Transfer the strain to PDB medium, culture it on a shaker at 30°C for 3 days, make a bacterial suspension, and then inoculate it in the fermentation medium with an inoculum amount of 5%, and place it in a constant temperature shaking culture at 30°C and 200r/min After culturing in the box for 5 days, the fermentation broth was centrifuged at 6000r/min for 10min at 4°C, and the supernatant was the crude enzyme solution. The content of reducing sugar in the crude enzyme solution was determined by DNS colorimetry, and the absorbance value was measured at 540nm with a microplate reader (Infinite M200Pro). The strains obtained by re-screening were inserted into PDA medium for repurification, and finally inoculated on PDA slant medium, and stored at 4°C until further identification of strains.

The strains with high filter paper weight loss rate and high enzyme activity were screened from the isolated strains, and the isolated strains were cultured in a constant temperature shaking incubator, and then obtained by adding PDA medium for repurification. The conditions for culturing in a constant temperature shaking incubator are 30° C., 200 r/min. PDA medium is to weigh 200g of peeled potatoes, 20g of glucose, 20g of agar, the pH is natural, cut the potatoes into small cubes, boil them in water for about 30 minutes, filter them with four layers of gauze, add glucose and agar powder to the filtrate, Heat and stir with a glass rod until the glucose and agar powder are completely dissolved, and finally add ultrapure water to 1000mL, and sterilize with damp heat at 121°C for 30min.

Example 2

This embodiment is a verification example of the degradation ability of a cellulase-producing fungus Trichoderma A-8 according to the present invention, comprising the following steps:

The filter paper disintegration test was carried out with the screened strains to verify their degradation ability. Each test tube was divided into 7mL filter paper medium, put 50mg (1cm×6cm) filter paper (weighed after drying) as the enzymolysis medium, inoculated the strains of different forms on the filter paper, and inoculated in a constant temperature incubator at 30°C. cultivated in. The test tube showing that the filter paper was decomposed was diluted, and streak culture was carried out in CMC-Na medium. After culturing at 30°C for 72 hours, the grown single colony was inoculated on the filter paper medium and cultured for 10 days. The filter paper was collected by filtration, washed with ultrapure water, dried at 80°C and weighed.

Gained bacterial strain is carried out the mensuration of cellulase activity:

The strains with large transparent circle, high filter paper weight loss rate and high enzyme activity were obtained by screening. Transfer the strains to PDB medium, culture on a shaking table at 30°C for 3 days, make a bacterial suspension, and then inoculate them in the fermentation medium with an inoculum of 5%, and place them in a constant temperature shaking culture at 30°C and 200r/min After culturing in the box for 5 days, the fermentation broth was centrifuged at 6000r/min for 10min at 4°C, and the supernatant was the crude enzyme solution. The content of reducing sugar in the crude enzyme solution was determined by DNS colorimetry, and the absorbance value was measured at 540nm with a microplate reader (Infinite M200Pro). The strains obtained by re-screening were inserted into PDA medium for repurification, and finally inoculated on PDA slant medium, and stored at 4°C until further identification of strains.

Example 3

This example is the molecular identification of cellulase producing bacteria.

From the morphological characteristics of the strain on PDA medium, it was preliminarily judged that the strain was a fungus. The strain was cultured in PDB medium for 48 hours, and the bacteria were collected by centrifugation for grinding, and then the strain DNA was extracted according to the extraction method and steps on the fungal DNA extraction kit. The extracted DNA was used as a template, and a fragment of about 450-600bp was amplified using universal primers ITS4/ITS5. The target fragment was amplified using the PCR reaction system of a commercial kit (2×Taq PCR MasterMix, Solarbio) and the polymerase chain reaction (PCR) program. After amplification, 5 μL of PCR products were migrated in 1.0% agarose gel for verification, and the electrophoresis results were observed on the stain-free electrophoresis imaging system (CHEMIDOC XRS, Bio-Rad). The PCR products were sent to Guangzhou Sangong for sequencing, and SeqMan software was used to splice the sequences and cut off redundant head and tail sequences and primer sequences. After that, the spliced sequences were aligned in multiple comparisons with Clustal X, and finally the obtained sequences were analyzed by the National Center for Biotechnology Information ( http://www.ncbi.nlm.gov) BLAST-N search program to compare ITS gene sequence data with the GenBank database. The ITS gene sequence was arranged, and the phylogenetic tree was constructed using the Neighbor Joining method under the guidance of 1000x using Mega 6.0 software to determine the evolutionary status of the strains.

Claims (7)

1. A strain of cellulase-producing fungus Trichoderma A-8, characterized in that the strain is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation number is CGMCC No.20746. 2. a screening method for cellulase producing bacteria Trichoderma A-8, characterized in that, comprising the following steps: (1) Get the moldy bagasse sample and place it in the sample bottle, and culture it statically in the culture medium; (2) Gradient dilution of the obtained bacterial solution was carried out, coated on a CMC-Na solid plate, placed in a constant temperature incubator for cultivation, and then cultured by streaking on a slant plane, and a single colony was separated and purified according to the colony shape and color; (3) The purified bacterial strain obtained by separation is taken and inserted into the Congo red culture medium with a puncher to cultivate, and the bacterial strain with a larger transparent circle is isolated; (4) Screen strains with high filter paper weight loss rate and high enzyme activity from the strains isolated in step (3), then place the screened strains in a constant temperature shaking incubator for cultivation, and then insert PDA medium for repurification get. 3. the screening method of a strain cellulase producing bacteria Trichoderma A-8 according to claim 2, is characterized in that, the substratum of described static culture is to be the liquid enrichment culture of carbon source with CMC-Na base. 4 . The method for screening a strain of cellulase-producing fungus Trichoderma A-8 according to claim 2 , wherein the cultivation in the constant temperature incubator is carried out at 30° C. for 5 days. 5. The screening method of a cellulase-producing fungus Trichoderma A-8 according to claim 2, wherein the Congo red culture medium is prepared according to the following ratio: (NH ₄ ) ₂ SO ₄ 2.0g, MgSO ₄ 7H ₂ O 0.5g, K ₂ HPO ₄ 1.0g, NaCl 0.5g, microcrystalline cellulose 2.0g, Congo red 0.4g, agar 20g, add ultrapure water to 1000mL; after preparation, 121℃ Moist heat sterilization for 30min. 6 . The method for screening a strain of cellulase-producing fungus Trichoderma A-8 according to claim 2 , wherein the conditions for culturing in a constant temperature shaking incubator are 30° C. and 200 r/min. 7. the screening method of a cellulase producing bacteria Trichoderma A-8 according to claim 2, is characterized in that, described PDA substratum is to take by weighing peeled potato 200g, glucose 20g, agar 20g, The pH is natural, the potatoes are cut into small cubes, boiled in water for about 30 minutes, and then filtered with four layers of gauze, adding glucose and agar powder to the filtrate, heating, stirring with a glass rod until the glucose and agar powder are completely dissolved, and finally adding ultra-pure Water to 1000mL, sterilized by moist heat at 121°C for 30min.

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