CN114317325A - Mangrove forest bacillus and its application and straw cellulose degradation method - Google Patents

Abstract

The invention discloses a mangrove bacillus, the strain preservation number of the mangrove bacillus is CGMCC 23765, and the gene sequence of the 16s rDNA of the mangrove bacillus is shown in SEQ ID No. 1 of the sequence table. The mangrove bacillus is applied to the degradation of cellulose of straw. The degradation method is as follows: spread the mangrove bacillus strain on the LB solid plate, and cultivate for 2 days; use sterile water to make the cultured mangrove bacillus strain into a bacterial suspension; respectively take 5 mL of the bacterial suspension to inoculate the bacteria suspension containing In the medium of straw, and shake culture at 28 °C and 120 rpm; after 10 d, remove the residual straw by filtration, rinse with distilled water and dilute hydrochloric acid solution to remove inorganic salts, as well as bacteria and CaCO ₃ precipitation attached to the straw; After drying at 80°C to constant weight, the straw degradation rate was calculated by weight loss method. The mangrove bacillus of the invention has the advantages of high heat-resistance ability, fast inducing enzyme production speed and strong degrading ability, etc., and is of great significance for the development of agricultural straw degrading inoculants.

Description

Mangrove bacillus and its application and straw cellulose degradation method

technical field

The invention relates to the technical field of microorganisms, in particular to a mangrove bacillus and its application and a method for degrading straw cellulose.

Background technique

Cellulose is the most abundant renewable resource on earth. After being metabolized by microorganisms, it can be converted into energy, feed and chemical raw materials. It is also one of the important links in the carbon cycle of the ecosystem. There are many fungi and bacteria that can degrade cellulose in nature, and they can secrete a variety of cellulases, such as β-glucanase, endoglucanase, exoglucanase, xylanase, Mannanase, peroxidase and esterase, etc., and have been industrially applied in many fields such as food, feed, medicine, textile, detergent and paper industry.

In addition, agricultural planting produces a large amount of straw waste. With the implementation of the policy to ban straw burning, new environmentally friendly methods are needed to deal with these straw wastes. Compared with chemical methods, biological treatment methods based on cellulase or cellulose degrading bacteria cause less pollution to the environment. However, due to the large amount of enzymes and high cost of microbial methods, it is currently difficult to be widely used in the treatment of straw waste. Therefore, it is still necessary to screen cellulose-degrading bacteria with high yield, strong decomposition ability and wide adaptability from special ecological environments, thereby reducing the cost of industrial application.

Mangroves are a unique woody plant community in the intertidal zone of the Gulf estuary where tropical and subtropical land and sea meet. Because the soil in the mangrove area contains a large amount of macromolecular organic matter such as cellulose, lignin and chitin, many microorganisms in the mangrove soil, especially fungi and actinomycetes, can produce various cellulose-degrading enzymes. Therefore, the mangrove ecological environment is an important resource bank of microbial strains. Screening high-efficiency cellulose-degrading bacteria from the mangrove ecological environment has high research and industrial application value.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for degrading straw cellulose by using mangrove bacillus, so as to solve the technical problems in the prior art.

In order to solve the above-mentioned technical problems, the present invention specifically provides the following technical solutions:

A mangrove bacillus, the strain deposit number of the mangrove bacillus is CGMCC23765, and the gene sequence of the 16s rDNA of the mangrove bacillus is shown in SEQ ID No. 1 of the sequence table.

As a preferred solution of the present invention, after the mangrove bacillus is cultured on the medium for 48 hours, the H17 colony is round, milky white raised, the edges are neat and smooth, and the Gram staining is negative.

As a preferred solution of the present invention, after the mangrove bacillus is cultured on the cellulose medium for two days, the Congo red staining shows that the diameter of the hydrolysis circle (D) and the diameter of the colony (d) are 13.70±2.33 mm, 4.30±2.3 mm, respectively. 0.67mm; D/d value 3.19.

The invention also provides the application of mangrove bacillus, which is applied to the degradation of cellulose of straw.

As a preferred solution of the present invention, the straw includes wheat straw, corn straw, rape straw and rice straw.

The present invention also provides a method for degrading straw cellulose by utilizing the above-mentioned mangrove bacillus, comprising the following steps:

Step 100, coating the mangrove bacillus strain on the LB solid plate or inoculating it into the LB liquid medium, and culturing for 2d;

Step 200, with sterile water, the mangrove bacillus strain after the cultivation is made into bacterial suspension;

Step 300, respectively taking 5 mL of the bacterial suspension and inoculating it into a liquid medium containing straw, and shaking culture at 28° C. and 120 rpm;

After steps 400 and 10d, the residual straws are filtered out, rinsed with distilled water and dilute hydrochloric acid solution to remove inorganic salts, as well as bacterial cells and CaCO3 precipitation attached to the straws _;

Steps 500 and 80°C are dried to constant weight, and the straw degradation rate is calculated by the weight loss method.

As a preferred solution of the present invention, the calculation formula of the straw degradation rate is:

Straw degradation rate%=(W ₀ -W _t )/W ₀ ×100%;

Among them, W ₀ represents the initial dry weight of straw; W _t represents the dry weight of straw after t days of culture.

As a preferred solution of the present invention, the composition of the straw degradation medium is: 1.0g/LKH ₂ PO ₄ , 0.1g/L NaCl, 0.3g/L MgSO ₄ ·7H ₂ O, 2.5g/L NaNO ₃ , 0.1g/L CaCl ₂ , 0.01g/L FeCl ₃ , and distilled water to 1000mL.

Compared with the prior art, the present invention has the following beneficial effects:

The mangrove bacillus of the invention has the advantages of high heat resistance, fast inducing enzyme production speed and strong degradation ability, is green and environmentally friendly, and is environmentally friendly. doses are important.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other implementation drawings can also be obtained according to the extension of the drawings provided without creative efforts.

Fig. 1 is the colony morphology (a) and Gram staining result (b) of Mangrove bacillus of the present invention;

Fig. 2 is the Congo red staining result of mangrove bacillus of the present invention;

Fig. 3 is a phylogenetic tree of the 16srDNA sequence of the mangrove bacillus of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the present invention, the mangrove bacillus with the deposit number CGMCC23765 is mainly collected from mangrove environmental samples, and the gene sequence of the 16s rDNA of the mangrove bacillus is shown in SEQ ID No. 1 of the sequence table.

The characteristics of mangrove bacillus after 48h culture on the medium are as follows:

H17 colonies were round, milky white raised, neat and smooth edges, and Gram staining was negative. After the mangrove bacillus was stained with Congo red (as shown in Figure 2), the diameter of the hydrolysis circle (D) and the diameter of the colony (d) were 13.70±2.33mm and 4.30±0.67mm, respectively; the D/d value was 3.19.

The invention also provides the application of mangrove bacillus, which is applied to the degradation of cellulose of straw.

As a preferred solution of the present invention, the straw includes wheat straw, corn straw, rape straw and rice straw.

The present invention also provides a method for degrading straw cellulose by utilizing the above-mentioned mangrove bacillus, comprising the following steps:

Step 100, the mangrove bacillus strain is coated on the LB solid plate or inoculated on the liquid medium, cultivated for 2d;

Step 200, with sterile water, the mangrove bacillus strain after the cultivation is made into bacterial suspension;

Step 300, respectively taking 5 mL of the bacterial suspension and inoculating it into the degradation medium containing straw, and shaking culture at 28°C and 120rpm;

After steps 400 and 10d, the filtered straws are taken out, rinsed with distilled water and dilute hydrochloric acid solution to remove inorganic salts, as well as bacterial cells and CaCO3 precipitation attached to the straws;

Steps 500 and 80°C are dried to constant weight, and the straw degradation rate is calculated by the weight loss method.

As a preferred solution of the present invention, the composition of the straw degradation medium is: 1.0g/L KH ₂ PO ₄ , 0.1g/L NaCl, 0.3g/L MgSO ₄ ·7H ₂ O, 2.5g/L NaNO ₃ , 0.1g/L CaCl ₂ , 0.01g/L FeCl ₃ , and distilled water to dilute to 1000mL.

Hereinafter, the present invention will be described in more detail by way of examples, but it is not intended to limit the present invention.

Example 1: Screening of cellulose degrading strains

(1) On April 28, 2021, environmental samples of mangroves in Binjiang Wetland Park in Foshan City were collected. 5-10 g of soil within 10 cm of the surface was collected from each site, a total of 5 sample points were collected, put into sterile bags, and stored in ice packs.

(2) Take 10 g of soil samples into a 250 mL conical flask, add 90 mL of sterile water under aseptic conditions, and shake at 200 rpm and 28 °C for 2 h.

Take 10 mL of the sample stock solution, and use the dilution plate coating method to dilute the stock solution to 10 ^-6 with a 10-fold gradient. Take 100 μL of each gradient and evenly spread it on the solid medium plate. After standing, transfer to 28 °C for constant temperature incubation Inverted culture in the box.

After the colonies grow, add an appropriate amount of 1g/L Congo red staining solution for staining (as shown in Figure 2) for 1 hour, then rinse with 1mol/L normal saline, select a single colony with a clear transparent circle around the colony, and use the continuous streaking method. Repeated streaking and purifying culture on CMC solid medium until pure colonies were obtained, and the purified strains were numbered and preserved.

Pick a single colony on the plate with a sterile toothpick and inoculate it on the CMC solid medium containing 1 g/L Congo red. After the colony grows, observe the transparent circle on the plate, and use a vernier caliper to measure the colony diameter (d) and the diameter of the transparent circle. (D).

The ratio (D/d) of the diameter of the transparent circle to the diameter of the colony was calculated, and the strains with stronger cellulose degrading ability were screened out according to the ratio.

Conclusion: It is found that H17 strain has strong cellulose degradation ability, and further research is planned.

Example 2: Molecular biological identification of H17 strain

(1) The H17 strain was inoculated into 200 mL of LB liquid culture medium, and cultured with shaking at 180 rpm and 37 °C for 48 h.

150 μL of bacterial solution was taken, and genomic DNA was extracted by bacterial genomic DNA extraction kit (Hangzhou Biori).

Using DNA as a template, PCR amplification of 16S rDNA was carried out using bacterial universal primers. The primer sequences were: upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

The PCR reaction system was 25 μL: template DNA 2 μL, upstream and downstream primers (10 μmol/L) 1 μL each, Taq mix 12.5 μL, and ddH ₂ O 8.5 μL.

Among them, PCR amplification conditions: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1.5 min, 30 cycles; extension at 72 °C for 10 min.

The PCR products were detected by 1.2% agarose gel electrophoresis, and the qualified PCR products were sent to BGI for sequencing.

(2) The sequencing results of H17 strains were compared and analyzed by BLAST in the NCBI database. Using MEGA 7.0 software, a phylogenetic tree was constructed by Neighbor-Joining (as shown in Figure 3). The results of 16S rDNA sequencing showed that the H17 strain was most closely related to Mangrovebacter plantisponsor strain k182, with a similarity of 98.89%, and the colony morphology and Gram staining results were also consistent with it, as shown in Figure 1.

Therefore, it was confirmed that the H17 strain was a strain of Mangrove bacillus.

Example 3: Determination of enzyme activity of H17 strain

(1) The H17 strain was inoculated into LB liquid medium and cultured with shaking at 28°C for 48h.

Then take 1 mL of bacterial liquid from it and inoculate it into 200 mL of LB liquid medium, conduct shaking culture at 34, 36, 38, 40 and 42 °C respectively, and take 4 mL of culture solution at 0, 4, 8, 12, 16 and 20 h, respectively. The collected bacterial liquid was centrifuged at 8000 rpm for 10 min at 4°C, and the obtained supernatant was the crude enzyme liquid.

(2) Prepare 1mg/mL glucose mother solution, respectively take 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0mL in a clean colorimetric tube, and make up 2mL with ultrapure water, then Add 2.5mL of DNS solution and shake well, take a boiling water bath for 5min, cool quickly and dilute to 10mL with distilled water, and mix well.

Take 100 μL and use a microplate reader to measure the OD value at a wavelength of 540 nm, take the OD value measured at 540 nm as the ordinate, and draw the standard curve with the glucose content (mg) as the abscissa to obtain the linear equation y=0.7815x-0.0006, R2 =0.9982.

(3) Take 4 clean test tubes with the same specifications, 1 is blank, and the other 3 are tubes to be tested. Accurately measure 1.5mL of 1% CMC-Na standard solution, add them to 4 test tubes respectively, add 0.5mL of crude enzyme solution to each of the 3 test tubes, shake well and place the 4 test tubes in a 50°C water bath. The reaction was carried out for 30 minutes.

After taking it out, add 0.5 mL of boiling inactivated crude enzyme solution to the blank tube, shake it well, immediately add 1.5 mL of DNS reagent to each test tube, take a boiling water bath for 10 minutes, take out and quickly cool to room temperature, and add distilled water to each test tube respectively Dilute to 10mL, shake well and let stand.

The OD value was measured at 540 nm, the reducing sugar content was determined according to the glucose standard curve, and the carboxymethyl cellulase activity was then calculated.

(4) At 16 h, the enzyme activity of H17 strain was 36℃>38℃>40℃>34℃>42℃ compared with the temperature treatment groups.

Example 4: Determination of Mangrove Bacillus H17 strain to degrade straw

(1) The H17 strain was spread on the LB solid plate, and after culturing for 2 days, a bacterial suspension was made with sterile water, and 5 mL of the bacterial suspension was inoculated into the degradation medium containing straw, and the culture was shaken at 28°C and 120 rpm. .

The composition of straw degradation medium is: 1.0g/L KH ₂ PO ₄ , 0.1g/L NaCl, 0.3g/L MgSO ₄ ·7H ₂ O, 2.5g/L NaNO ₃ , 0.1g/L CaCl ₂ , 0.01 g/L FeCl ₃ , dilute to 1000 mL with distilled water.

The straws used are from wheat, corn, rape and rice, respectively.

After 10 days, the filtered straws were taken out, rinsed with distilled water and dilute hydrochloric _acid solution to remove inorganic salts, bacteria attached to the straws and CaCO3 precipitation, etc., dried at 80 °C to constant weight, and the straw degradation rate was calculated by the weight loss method.

Straw degradation rate%=(W ₀ -W _t )/W0×100%;

Among them, W ₀ represents the initial dry weight of straw; W _t represents the dry weight of straw after t days of culture.

(2) After culturing for 10 days, strain H17 showed certain degradation ability to wheat, corn, rapeseed and rice straw, and the degradation rates of 10 days were 5.8%, 13.5%, 9.5% and 12.4%, respectively.

To sum up, the mangrove bacillus of the present invention has the advantages of high heat resistance, fast inducible enzyme production speed and strong degradation ability, is green and environmentally friendly, and is environmentally friendly. Straw degrading bacteria are of great significance.

The above embodiments are only exemplary embodiments of the present application, and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements should also be regarded as falling within the protection scope of the present application.

sequence listing

<110> Anhui Normal University

<120> Mangrove bacillus and its application and straw cellulose degradation method

<141> 2021-12-01

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1444

<212> DNA

<213> Mangrovibacter plantisponsor

<400> 1

cagcttggcg cagctacaca tgcagtcgag cggcagcggg aagaagcttg cttctttgcc 60

ggcgagcggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg ggataactac 120

tggaaacggt agctaatacc gcataacgtc gcaagaccaa agagggggac cttcgggcct 180

cttgccatcg gatgtgccca gatgggatta gctggttggt gaggtaacgg ctcaccaagg 240

cgacgatccc tagctggtct gagaggatga ccagccacac tggaactgag acacggtcca 300

gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc 360

atgccgcgtg tatgaagaag gccttcgggt tgtaaagtac tttcagtcag gaggaaggtg 420

gtgaacttaa tacgttcatc aattgacgtt actgacagaa gaagcaccgg ctaactccgt 480

gccagcagcc gcggtaatac ggagggtgca agcgttaatc ggaattactg ggcgtaaagc 540

gcacgcaggc ggtctgtcaa gtcggatgtg aaatccccgg gctcaacctg ggaactgcat 600

tcgaaactgg caggctagag tctcgtagag ggaggtagaa ttccaggtgt agcggtgaaa 660

tgcgtagaga tctggaggaa taccggtggc gaaggcggcc tcctggacga agactgacgc 720

tcaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780

cgatgtcgac ttggaggctg tgcccttgag gcgtggcttc cggagctaac gcgttaagtc 840

gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac 900

aagcggtgga gcatgtggtt taattcgatg caacgcgaag aaccttacct ggtcttgaca 960

tccagagaat cctgcagaga tgcgggagtg ccttcgggaa ctctgagaca ggtgctgcat 1020

ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080

atcctttgtt gccagcggtt aggccgggaa ctcaaaggag actgccagtg ataaactgga 1140

ggaaggtggg gatgacgtca agtcatcatg gcccttacga ccagggctac acacgtgcta 1200

caatggcgca tacaaagaga agcgaacttg cgagagtaag cggacctcat aaagtgcgtc 1260

gtagtccgga ttggagtctg caactcgact ccatgaagtc ggaatcgcta gtaatcgcgg 1320

atcagaatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1380

gagtgggttg caaaagaagt aggtagctta accttcggga gggcgctacc actttggatc 1440

aagg 1444

Claims (8)

1. a mangrove bacillus, is characterized in that, the bacterial classification deposit number of described mangrove bacillus is CGMCC23765, and the gene sequence of the 16s rDNA of described mangrove bacillus is as shown in sequence table SEQ ID No.1. 2. a kind of mangrove bacillus according to claim 1, is characterized in that: after described mangrove bacillus is cultivated on medium for 48h, H17 bacterium colony is round, milky white bulge, edge are neat and smooth, and Gram staining is feminine. 3. a kind of mangrove bacillus according to claim 1 is characterized in that: described mangrove bacillus is cultivated on cellulose medium after two days, shows hydrolysis circle diameter (D) and colony diameter (d) through Congo red staining. ) were 13.70±2.33mm and 4.30±0.67mm, respectively; the D/d value was 3.19. 4. An application of mangrove bacillus, characterized in that, it is applied to the degradation of cellulose of straw. 5 . The application of a mangrove bacillus according to claim 4 , wherein the straw comprises wheat straw, corn straw, rape straw and rice straw. 6 . 6. a straw cellulose degradation method utilizing the mangrove bacillus described in any one of claim 1-3, is characterized in that, comprises the steps: Step 100, coating the mangrove bacillus strain on the LB solid plate or inoculating it into the LB liquid culture solution, and culturing for 2d; Step 200, with sterile water, the mangrove bacillus strain after the cultivation is made into bacterial suspension; Step 300, respectively taking 5 mL of the bacterial suspension and inoculating it into a liquid culture medium containing straw, and culturing with shaking at 28° C. and 120 rpm; After steps 400 and 10d, the residual straws are taken out by filtration, rinsed with distilled water and dilute hydrochloric acid solution to remove inorganic salts, as well as bacteria and CaCO3 precipitation attached to the straws _; Steps 500 and 80°C are dried to constant weight, and the straw degradation rate is calculated by the weight loss method. 7. The method for degrading straw cellulose according to claim 6, wherein the calculation formula of the straw degradation rate is: Straw degradation rate%=(W ₀ -W _t )/W ₀ ×100%; Among them, W ₀ represents the initial dry weight of straw; W _t represents the dry weight of straw after t days of culture. 8 . The method for degrading straw cellulose according to claim 6 , wherein the composition of the straw degrading medium is: 1.0 g/L KH ₂ PO ₄ , 0.1 g/L NaCl, 0.3 g/L MgSO ₄ ·7H ₂ O, 2.5 g/L NaNO ₃ , 0.1 g/LCaCl ₂ , 0.01 g/L FeCl ₃ , and distilled water to make up the volume to 1000 mL.

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