High-temperature-resistant cellulose degrading bacterium and application thereof

Abstract

The invention provides a high-temperature-resistant cellulose degrading bacterium and application thereof, belongs to bacillus (Bacillus), and is named as Bacillus licheniformis C1. The high-temperature-resistant cellulose degrading bacterium has the advantages that the high-temperature-resistant cellulose degrading bacterium can be used for degrading high-temperature-resistant cellulose; according to the invention, continuous enrichment culture is carried out in a cellulose degradation culture medium under a high-temperature condition, a high-temperature-resistant cellulose degradation bacterium C1 is separated, and the strain C1 is a species of bacillus through preliminary identification of multiphase taxonomy. The filter paper enzyme activity of C1 obtained through screening at the temperature of 50 DEG C reaches 14.014 + / -0.438 U / ml, the endo-cellulase activity reaches 1.664 + / -0.225 U / ml, the exo-cellulase activity reaches 3.909 + / -0.374 U / ml, and the C1 can play a role in lignocellulose treatment and fertilizer preparation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a high temperature resistant cellulose degrading bacterium and an application thereof. Background Art

[0002] Straw composting and fermentation is a complex physiological metabolic process that requires the synergistic participation of many microorganisms. Under different conditions, the substrate lignin is decomposed and utilized to different degrees, and the resulting compost products are also different. Incomplete composting will lead to a series of problems such as insufficient fertility of the product, excessive intermediate by-products, accumulation of toxic substances, excessive harmful and malodorous gases, etc., which have a great impact on resource utilization and environmental protection. In order to solve the problems in traditional straw composting, high-temperature aerobic composting technology has attracted more and more attention. In recent years, research on high-temperature aerobic composting and fermentation technology has found that straw composting with the participation of thermotolerant bacteria can significantly increase the compost heating rate, shorten the composting time, further decompose lignin, and improve the fertilizer efficiency of fertilizer products. Summary of the invention

[0003] The object of the present invention is to solve the defects of the above-mentioned prior art and provide a high-temperature resistant cellulose degrading bacterium and its application. The high-temperature resistant cellulose degrading bacterium C1 (Bacillus licheniformis) belongs to the genus Bacillus and is applied to straw composting through a composting process to accelerate the degradation of lignin and improve the quality of compost products.

[0004] The present invention adopts the following technical solution:

[0005] A high-temperature resistant cellulose-degrading bacterium was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 6, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. It was named Bacillus licheniformis C1 and the collection number is GDMCC NO: 65107.

[0006] The fermentation broth or culture broth of the aforementioned high temperature resistant cellulose degrading bacteria.

[0007] Application of thermostable cellulose degrading bacteria or fermentation liquid or culture liquid of the thermostable cellulose degrading bacteria in cellulose degradation.

[0008] In the application of the aforementioned high temperature resistant cellulose degrading bacteria in degrading cellulose, the cellulose refers to the cellulose in the straw in the biocompost.

[0009] Beneficial effects of the present invention:

[0010] The high temperature resistant cellulose degrading bacteria of the present invention can still maintain a high cellulase activity at a high temperature of 50°C and can degrade lignocellulose, so it can be widely used in the pretreatment of lignocellulose-containing materials or straw composting applications. Under high temperature conditions, the present invention isolates a high-tolerance cellulose degrading bacteria C1 by continuous enrichment culture of cellulose degradation medium, and the filter paper enzyme activity reaches 14.014±0.438U / ml, the endocellulase activity reaches 1.664±0.225U / ml, and the exocellulase activity reaches 3.909±0.374U / ml at a temperature of 50°C, which can play a role in lignocellulose treatment and fertilizer preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a Congo red staining image of thermostable cellulose-degrading bacteria.

[0012] Figure 2 This is the result of optical microscope observation of high temperature resistant cellulose degrading bacteria.

[0013] Figure 3 This is a phylogenetic tree of strains based on 16SrDNA constructed using the NJ method.

[0014] Figure 4 This is the growth of thermostable cellulose-degrading bacteria on pig manure extract plates.

[0015] Figure 5 This is the growth curve of thermostable cellulose-degrading bacteria.

[0016] Figure 6 This is a diagram of compost cellulose degradation.

[0017] Figure 7 This is a diagram of compost hemicellulose degradation.

[0018] Figure 8 This is a diagram of compost lignin degradation. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1 of the present invention: This embodiment provides a high-temperature resistant cellulose degrading bacteria, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 6, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, named Bacillus licheniformis C1, and the storage number is GDMCC NO: 65107.

[0021] This microorganism was found and isolated from samples of a composting plant in Hongya County, Meishan City, Sichuan Province.

[0022] The high temperature resistant cellulose degrading bacteria are mainly used to degrade cellulose in straw in high temperature composting.

[0023] In order to verify the performance of the thermostable cellulose degrading bacteria, the following is the experimental content:

[0024] 1. Materials and Methods

[0025] Source of strain: Compost sample from a chicken manure composting plant in Meishan City, Sichuan Province.

[0026] Reagents and culture medium: Filter paper, carboxymethyl cellulose (CMC), and microcrystalline cellulose were produced by Shanghai MacLean Biochemical Technology Co., Ltd.

[0027] Enrichment culture medium: (NH4)2SO45.0g / L, Na2HPO42.0g / L, K2HPO42.0g / L, NaCl 2.0g / L, MgSO4 0.5g / L, straw chips 15.0g / L, 121℃15min.

[0028] LB medium, beef extract peptone medium.

[0029] Cellulase verification culture medium: (NH4)2SO42.0g / L, MgSO40.5g / L, K2HPO41.0g / L, NaCl 0.5g / L, sodium carboxymethyl cellulose 15g / L, Congo red 0.4g / L, agar 15.0-20.0g / L, pH7-7.5.

[0030] Isolation of thermostable cellulose-degrading bacteria:

[0031] Take 5.0g of the sample and add it to 250ml of sterilized enrichment medium, place it in a 50℃ constant temperature shaker, 150rpm / min, and shake and culture for 48h. After the obtained enrichment culture solution is allowed to settle, 5.0mL of the upper bacterial liquid is taken and added to 250mL of new enrichment medium, and cultured under the same conditions for 48h. Repeat this step 5 times.

[0032] Dilution and spreading on plates: Take 1 mL of the culture medium obtained from the last enrichment, dilute it 10 times with sterile saline, take 100 μL of each gradient of bacterial solution dilution and spread it evenly on the LB plate, place it in a 50℃ constant temperature incubator and culture it upside down for 48-72 hours, and observe until a single colony appears.

[0033] Plate streak purification: In the clean bench, use a sterile inoculation loop to pick up single colonies with good growth, different colors and shapes, streak them on the LB solid plate, and invert them in a 50℃ constant temperature incubator for 48-72h. Repeat this purification step 3 times until a single colony with the same morphology and color is obtained. The purified single colony is streaked on the slant LB medium and stored in a refrigerated place at 4℃ for later use.

[0034] Functional verification of thermostable cellulose degradation strains: First, activate the strains and prepare seed solution. Take 100 μL of the obtained functional strain seed solution and drop it on the corresponding verification culture medium plate, culture it in a 50°C constant temperature incubator for 24-48 hours, and observe the growth of the colonies and the size of the transparent hydrolysis circle. Select single colonies with good growth and obvious hydrolysis apertures, and use the slant preservation method to store them for later use.

[0035] Cellulase activity assay:

[0036] All strains needed to be activated before enzyme activity determination. Use a sterile inoculation loop to pick a single colony of the strain with good growth from the preserved slant, inoculate it into beef extract peptone liquid culture medium, place it at 50°C, 180 rpm / min shaker, shake and culture for 24 hours to prepare seed solution.

[0037] Cellulase activity (filter paper enzyme, endocellulase, exocellulase) was determined by DNS method. Enzyme activity definition: At 50°C, the amount of enzyme required for 1.0 mL of crude enzyme solution to hydrolyze the substrate to produce 1 μg of glucose within 1 min is 1 cellulase activity unit (U / mL).

[0038] Pig manure extract growth verification: Since the screened strains will be used in pig manure composting production, pig manure extract growth verification is carried out on them. First, the required verification strains are activated and seed liquid is prepared. Take 1mL of the strain seed liquid, add 9mL of sterile water to make the starting bacterial liquid, and then dilute it appropriately. Take 1mL of the diluted bacterial liquid and spread it on the corresponding pig manure extract medium plate, culture it in a 50℃ constant temperature incubator for 24-48h, and observe the growth of the colony.

[0039] Strain growth curve and optimal growth temperature determination

[0040] First, activate the strain to be tested and prepare the seed solution. Dilute the seed solution with sterile physiological saline and adjust the OD600 to 0.800-1.000. Inoculate the diluted bacterial solution into 50mL of the corresponding liquid culture medium at a ratio of 2% (v / v), and culture it in a 50℃ constant temperature incubator for 48h. Sample once at the initial 0h, 4h, and 8h, and then sample once every 8h. Use an ultraviolet spectrophotometer to measure the OD value of the bacterial solution at a wavelength of 600nm, and draw a growth curve.

[0041] The above strain dilution was inoculated into 50mL LB medium at a ratio of 2% (v / v), five bottles each. It was placed in a constant temperature shaking shaker at 20℃, 30℃, 40℃, 50℃, and 60℃ for 12h, and the OD value of the bacterial solution at a wavelength of 600nm was measured using an ultraviolet spectrophotometer to obtain the growth of the strain at different temperatures.

[0042] Strain identification

[0043] The isolated strains were subjected to morphological observation, physiological and biochemical experiments, and 16rRNA gene sequence analysis.

[0044] Compost Application

[0045] Pig manure was selected as the material, and wheat straw was selected as the auxiliary material. Under laboratory conditions, a 60-80L insulation foam box was used for composting. A total of four treatments were set up. The composting time was set to 50 days. The material selection was pig manure: straw 3:1, water content 65±1%, C / N=21.5. For each treatment, 15.0kg of fresh pig manure and 5.0kg of corn straw were weighed and fully mixed as compost materials.

[0046] (1) Control CK: blank treatment.

[0047] (2) T1 treatment: 5% (by mass) C1 bacterial solution was applied, with a viable bacterial count of 8.10 6 (CFU / ml).

[0048] Aeration is achieved by connecting the air pump hose to the bottom of the foam box, and the ventilation inlet is connected to a gas flow meter, while ensuring the air tightness of the hose connection. The aeration volume is controlled at 0.4L / min·kg dry matter, which is converted to 3.2L / min.

[0049] Determination of cellulose content of samples before and after composting:

[0050] Use an analytical balance to accurately weigh 1.000g (recorded as m) of the sample and put it into a 50mL conical flask. Add 70mL of distilled water and mix well, then put it in a boiling water bath and heat it for 1 hour and take it out. After filtering through a G3 sand core funnel, rinse it with 95% ethanol, anhydrous ethanol and acetone in sequence, dry the filter residue at 80°C to constant weight, and record the data as m1. Put the above-mentioned dried filter residue into a crucible, add 70mL of 2mol / L HCl solution and mix it evenly, put it in a boiling steam pot and heat it for 50 minutes, and then filter it through a G3 sand core funnel again. Then rinse it with 95% ethanol, anhydrous ethanol and acetone respectively, dry the filter residue at 80°C to constant weight, and record the data as m2. Put the above-mentioned dried filter residue into a crucible, add 10mL of 72% sulfuric acid, react for 4 hours, add 90mL of distilled water and shake it evenly, and leave it at room temperature for 12 hours. Then wash the filter residue with distilled water and dry it at 80℃ to constant weight, record data m3. Place the dried filter residue in a muffle furnace at 550℃ and burn it to constant weight, record data m4. The final content of each component is calculated as follows:

[0051]

[0052] Results and Analysis

[0053] Isolation of thermostable cellulose-degrading bacteria

[0054] The cellulose-degrading strains were isolated and screened, and the degradation function was verified by Congo red staining to obtain the hydrolysis circle, such as Figure 1 Its growth was verified by culturing pig manure extract, as shown in Figure 4 shown.

[0055] Identification of thermostable cellulose-degrading bacteria

[0056] The strain of thermostable cellulose-degrading bacteria is a Gram-positive bacterium, facultative anaerobic and mobile. The colonies on the TSA plate are off-white, opaque, with smooth edges and slightly rough surfaces. The strain is rod-shaped, with a size of (0.3-0.6)×(1.5-3.5)μm. The growth temperature is 10-70℃, the pH range is: 5-9, and the tolerance to NaCl concentration is 8% (W / V). Glucose, sucrose, xylose, fructose, lactose, glycogen, glycerol, ribose, inositol, mannitol, raffinose, and maltose can be used as carbon sources. Some physiological and biochemical experimental results of strain C1 are shown in Table 1.

[0057] Table 1 Physiological and biochemical results of thermostable cellulose-degrading bacteria

[0058]

[0059] Through 16sRNA sequencing, the strain thermostable cellulose degrading bacteria was compared and analyzed on the Eztaxte website. The results showed that the strain thermostable cellulose degrading bacteria had a highest similarity of 99.37% with Bacillus licheniformis NCDO 1772. The phylogenetic tree was constructed, such as Figure 3 The results showed that the thermostable cellulose-degrading bacteria and Bacillus licheniformis NCDO1772 were in the same subclade, with high stability and close evolutionary relationship. The results of phylogenetic tree and sequence analysis showed that the thermostable cellulose-degrading bacteria belonged to the genus Bacillus.

[0060] Strain growth characteristics, enzyme activity determination: Figure 5 It shows that 8 hours before inoculation, the growth of the strain is in a slow phase; 8 hours later, the strain enters a logarithmic growth phase, the bacteria multiply rapidly, and the bacterial solution gradually becomes turbid; after 16 hours, the growth rate of the bacterial colony slows down; after 24 hours, the bacterial colony is basically stable, and OD600 reaches its maximum value; after 40 hours, the bacterial colony drops for the first time. The growth curve of the strain conforms to the S-shaped growth curve of the biological population, and K / 2 is reached in 8-12 hours. At this time, the growth, metabolism and reproduction rate of the bacterial colony is the highest, which should be considered as the best time for inoculation in later experiments. The strain was cultured at different temperatures for 12 hours, and the OD600 value was measured. The results are as follows Figure 2 As shown in the figure, the thermotolerant cellulose-degrading bacteria grew best at 50°C, and were significantly better than those at other temperatures.

[0061] The standard curve required for measuring cellulase activity is the glucose standard curve. The filter paper enzyme activity, endocellulase activity, and exocellulase activity of the strains measured by the DNS method are shown in Table 2.

[0062] Table 2 Cellulase activity of strains

[0063]

[0064] Application of thermotolerant cellulose-degrading bacteria in composting

[0065] The changes of various components of treated lignocellulose are as follows Figure 6-Figure 8As shown. From the overall trend of cellulose and hemicellulose, all treatments are in line with the fastest degradation rate in the first 10 days, and there is almost no significant data change after 10 days. It shows that the explanation of cellulose and hemicellulose is mainly concentrated in the composting warming period and high temperature period, and there is little change in the cooling period and mature stage. In terms of cellulose degradation, the content of CK and T1 decreased by 23.1% and 34.0%, respectively. The hemicellulose content of CK and T1 decreased by 24.8% and 37.5%, respectively. The two data show that T1 inoculated with microorganism C1 can significantly promote the degradation of cellulose composting. Lignin is the most difficult substance to degrade and utilize in lignocellulose. The figure shows that the lignin content of each treatment group showed an upward trend within 0-3d, and then gradually decreased. The reason for the analysis may be that the degradation process of lignin is very difficult and slow, and the rapid degradation of cellulose and hemicellulose in the early stage of composting leads to a relative increase in the lignin content. At the end of composting, the lignin degradation rates of CK and T1 were 11.4% and 20.9%, respectively. The lignin degradation rates of the processors inoculated with microorganisms were higher than that of the blank control, and the final content was also lower than that of the blank control group.

[0066] in conclusion

[0067] The strain of thermostable cellulose-degrading bacteria obtained by screening belongs to Bacillus licheniformis in Bacillus, which can withstand a high temperature environment of 50°C while maintaining a high activity of various types of lignocellulase. Through composting application experiments, it was found that the degradation effects of the three types of cellulose added with microbial thermostable cellulose-degrading bacteria were better than those of the CK group, and the effect was improved by 50% compared with the CK group. The thermostable cellulose-degrading bacteria can be applied to straw compost production.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thermostable cellulose-degrading bacterium, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 6, 2024, named Bacillus licheniformis C1, and the preservation number is GDMCCNO:65107.

2. A fermentation broth or culture broth containing the thermostable cellulose degrading bacteria according to claim 1.

3. Use of the fermentation liquid or culture liquid of the high temperature resistant cellulose degrading bacteria according to claim 1 or the high temperature resistant cellulose degrading bacteria according to claim 2 in degrading lignocellulose.

Images