Bacillus for degrading cellulose and application thereof

Abstract

The invention belongs to the technical field of microbial degradation of cellulose. The invention provides a bacillus sp. R22, and the preservation number of the bacillus sp. R22 is CGMCC (China General Microbiological Culture Collection Center) No. 27961. The strain R22 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 18, 2023, the center is called CGMCC for short, the address of the center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the name of the strain R22 is bacillus sp., and the preservation number of the strain R22 is CGMCC No.27961. The strain R22 is named as bacillus sp. The strain R22 provided by the invention has an obvious degradation effect on cellulose.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation of cellulose technology. Background Technology

[0002] Due to its renewable nature, cellulosic biomass is considered one of the most promising and sustainable natural resources for biofuels, and a raw material for developing useful organic compounds, feed additives, and other value-added products. Because of its wide distribution and abundance in nature, it can also serve as a substrate for producing various industrially significant enzymes. Cellulose is a highly valuable organic substance with abundant sources, occupying an irreplaceable and vital position on Earth.

[0003] Cellulose constitutes a significant portion of agricultural and forestry resources, but most of it is not utilized rationally and is instead burned or discarded, causing environmental pollution. Therefore, screening for superior bacteria capable of effectively degrading cellulose is crucial for its efficient conversion into smaller molecules or other nutrients. Summary of the Invention

[0004] The purpose of this invention is to provide a cellulose-degrading bacterium. This strain was isolated and screened from black soil samples in the Lishu-Maize-Soybean Black Soil Demonstration Area in Siping, Jilin Province. The strain exhibits significant cellulose degradation activity, providing a material basis for remediating cellulose pollution in soil.

[0005] Strain R22 was deposited on July 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The name of strain R22 is Bacillus sp., and the accession number of strain R22 is CGMCC No. 27961. Attached Figure Description

[0006] Figure 1 This is a colony morphology diagram of the novel cellulose-degrading bacteria of this invention.

[0007] Figure 2 This is an electron micrograph of the cell morphology of the novel cellulose-degrading bacteria of this invention.

[0008] Figure 3 This is a phylogenetic tree diagram of the novel cellulose-degrading bacteria and similar model strains of this invention.

[0009] Figure 4 This is a colony morphology diagram of Bacillus of the present invention.

[0010] Figure 5 This is the staining result of the strain on Congo red medium.

[0011] Figure 6 This is a diagram showing the results of a filter paper disintegration experiment. Detailed Implementation

[0012] Example

[0013] The strain R22 was isolated and screened from black soil samples in the Lishu-Maize-Soybean Black Soil Demonstration Area of ​​Siping City, Jilin Province. This strain exhibits significant cellulose degradation activity, providing a material basis for remediating cellulose pollution in soil. The 16S rDNA sequence of the cellulose-degrading bacterium is shown in SEQ ID NO.1.

[0014] Strain R22 was deposited on July 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The name of strain R22 is Bacillus sp., and the accession number of strain R22 is CGMCC No. 27961.

[0015] After being cultured on R2A solid medium for one day, the strain exhibited the following phenotypic characteristics: flat colonies with undulating edges, forming a honeycomb ring around the colony; dull and granular texture; white or cream-colored; single-celled rod-shaped cells; growth temperature of 20-40℃; and pH of 4-8. Under electron microscopy, the bacteria were rod-shaped, arranged singly or in pairs. Figure 1 and Figure 2 As shown.

[0016] The most sequence-similar type strain of the cellulose-degrading strains is: Bacillus mobilis0711P9-1 T The full-length similarity of the 16S sequence was 98.67%. A phylogenetic tree was constructed using the 16S sequence of similar Bacillus type strains; the phylogenetic tree is shown below. Figure 3 As shown, sequence similarity and evolutionary relationship confirm that it is a member of the genus Bacillus.

[0017] The enrichment medium (MSG) (1L) consisted of: potassium dihydrogen phosphate 0.5g, disodium hydrogen phosphate dodecahydrate 1g, magnesium sulfate heptahydrate 0.2g, ammonium chloride 1.1g, glucose 1g, cellulose 200mg, trace elements 2ml, vitamins 2ml, pH=7.3. The trace elements (1L) consisted of: dipotassium hydrogen phosphate 5g, magnesium sulfate 2.5g, sodium chloride 2.5g, ferrous sulfate 0.05g, and manganese sulfate 0.05g.

[0018] R2A solid medium (1L): yeast extract 0.5g, peptone 0.5g, casein amino acids 0.5g, glucose 0.5g, soluble starch 0.5g, dipotassium hydrogen phosphate 0.3g, magnesium sulfate heptahydrate 0.05g, sodium pyruvate 0.3g, agar 15g, pH=7.2.

[0019] Citrate buffer (0.05 mol / L, pH 4.8): Weigh 2.2065 g of citric acid and dissolve it in 105 ml of ultrapure water to obtain solution A; weigh 4.4115 g of citric acid and dissolve it in 150 ml of ultrapure water to obtain solution B; mix 102.5 ml of solution A and 147.5 ml of solution B thoroughly, add double-distilled water to a final volume of 500 ml, and adjust the pH appropriately to obtain the citrate buffer.

[0020] Congo red staining solution (1 mg / ml): Weigh 0.20 g of Congo red, add 500 ml of ultrapure water and stir to dissolve.

[0021] Congo red dye solution.

[0022] Sodium chloride aqueous solution (1 mol / L): Weigh 29.00 g of sodium chloride and add 500 ml of ultrapure water to prepare a 1 mol / L solution.

[0023] Sodium chloride aqueous solution.

[0024] Sodium carboxymethyl cellulose identification medium: 20g CMC-NA, 10g peptone, 10g yeast extract, 5g sodium chloride, 1g KH2PO4, 0.2g MgSO4, 1000mL distilled water, sterilized at 121℃ for 20min.

[0025] 1. Isolation and screening of cellulose-degrading bacteria R22

[0026] Weigh 5g of soil sample and place it in a 100ml sterile Erlenmeyer flask. Add 100ml of sterile water and incubate on a shaker at 30℃ and 180 rpm for 1 h. Inoculate 5ml of the soil suspension into a 100ml enrichment solution.

[0027] In the culture medium, the culture was enriched at 25℃ and 160 rpm for 3 days. Following a serial dilution method, 100 μl of each concentration was taken and spread onto selection medium, and incubated upside down at room temperature for 3 days. Strains with different colony morphologies were picked using an inoculation loop, streaked onto differential culture medium plates for isolation and purification, and then numbered and stored.

[0028] The above-mentioned microbial enrichment medium (MSG) (1L) consists of: potassium dihydrogen phosphate 0.5g, disodium hydrogen phosphate dodecahydrate 1g, magnesium sulfate heptahydrate 0.2g, ammonium chloride 1.1g, glucose 1g, cellulose 200mg, trace elements 2ml, vitamins 2ml, pH=7.3. Trace elements (1L): dipotassium hydrogen phosphate 5g, magnesium sulfate 2.5g, sodium chloride 2.5g, ferrous sulfate 0.05g, manganese sulfate 0.05g.

[0029] The purified strain was inoculated onto CMC agar medium and cultured at 37°C for 48 h. A suitable amount of Congo red chromogenic solution was added to a cellulose identification plate, submerging the CMC agar plate completely. The plate was then allowed to stand for 20 min, and the chromogenic solution was discarded. The plate surface was rinsed 2–3 times with NaCl solution (1 mol / L). The formation of the transparent zone was observed, and the diameter of the transparent zone and the colony diameter (D) and (D / d) on the plate were accurately measured using calipers. The ratio of the diameter to the colony diameter was calculated, and the petri dish was photographed using a mobile phone.

[0030] 2. Identification of cellulose-degrading bacteria R22

[0031] Phenotypic characteristics such as Figure 4 As shown: When strain R22 is inoculated onto R2A plates and grown at 30°C for 1 day, the colonies are flat with undulating edges and a honeycomb ring around them. They are dull and granular, with a white or creamy color. They are Gram-positive and grow at temperatures of 20-38°C and pH of 4-8.

[0032] 16S rDNA identification: Strain R22 was inoculated into R2A medium and cultured in a shaker at 30℃ for 1 day. 2 μl of the bacterial culture was used as a template for PCR amplification using universal bacterial primers 27F and 1492R. 2×Es Taq Master Mix premixed solution from Kangwei Century Biotechnology Co., Ltd. was used. The PCR reaction volume was 30 μl, specifically: 2 μl bacterial culture, 1 μl 27F primer, 1 μl 1492R primer, 15 μl 2×Es Taq Master Mix, and ddH2O. The reaction program was: 94℃ for 5 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 60 s, 30 cycles, 72℃ for 5 min. The purified PCR product was sent to Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The obtained DNA sequence was uploaded to the EZBioCloud database for sequence alignment, obtaining the known sequence with the highest similarity to the aligned sequence and the standard strain sequence. Analysis revealed that the highest sequence similarity was found to Bacillus mobilis, with a similarity of 99.67%. This indicates that the degrading bacteria isolated in this invention belong to the genus Bacillus mobilis.

[0033] 3. Results of Bacillus rescreening in this invention

[0034] After initial screening on the identification medium and staining with Congo red solution, strain R22 showed a distinct clear zone. Congo red is an anionic azo dye; its hydrophobic regions and sulfonic acid groups can tightly bind to the β-1,4-glycosidic bonds in cellulose molecules through hydrogen bonds and hydrophobic interactions, forming a stable "cellulose-Congo red complex." On agar plates containing Congo red and cellulose, cellulase is secreted around cellulose-degrading colonies. In the enzymatically hydrolyzed areas, cellulose is degraded, Congo red loses its binding sites and fades, forming a clear hydrolysis zone. The cellulose-Congo red complex in the non-degraded areas remains red. Figure 5 The clear zone on the medium plate confirms this conclusion, thus strain R22 has the ability to degrade cellulose.

[0035] 4. Filter paper strip disintegration test

[0036] The dominant strains identified by enzyme activity assays were inoculated into beef protein broth and cultured for 24 h to prepare bacterial suspensions. 1 mL of each bacterial suspension was inoculated into an Erlenmeyer flask containing 100 mL of sterile filter paper strip disintegration medium, and three sterile Waterman® filter paper strips were added. The flasks were incubated at 28℃ and 100 rpm, with periodic observation of filter paper strip disintegration. Each strain was replicated three times. The degree of filter paper strip disintegration was used to preliminarily determine the strain's ability to degrade cellulose.

[0037] Day 1 after inoculation with strain R22, the filter paper strips had completely decomposed into a paste, while the control group showed no obvious decomposition. Figure 6 As shown, the cellulose degradation ability of strain R22 can be determined.

Claims

1. Bacillus sp. R22, with accession number CGMCC No.27961.

2. A microbial inoculant containing Bacillus R22 as described in claim 1.

3. A composition comprising Bacillus R22 as claimed in claim 1.

4. The application of Bacillus R22 as described in claim 1 in the degradation of cellulose.

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