A high temperature resistant cellulose degrading bacterial agent and its preparation method and application

Abstract

The embodiment of the present application relates to a high temperature resistant cellulose degrading bacterial agent and its preparation method and application, which belongs to the field of biological environmental protection technology. The embodiment of the present application aims to solve the technical problems that crop straw is difficult to degrade, has a long decomposition period, and has poor product quality in traditional composting. The high temperature resistant cellulose degrading bacterial agent of the embodiment of the present application is characterized in that the high temperature resistant cellulose degrading bacterial agent is Bacillus thermoglucosidase paragenes R-7; the Bacillus thermoglucosidase paragenes R-7 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 1, 2024, with a deposit number of CGMCC No.30236, and the classification name is Parageobacillus thermoglucosidasius. The high temperature resistant cellulose degrading bacterial agent prepared by the method of the present application is applied in the mixed composting process of biogas slurry and straw, which can shorten the fertilizer time and improve the compost maturity. The addition of high temperature resistant cellulose degrading bacterial agent is beneficial to the degradation of cellulose and hemicellulose in the pile, and improves the quality of the compost product.

Description

Technical Field

[0001] The embodiments of the present application relate to the field of biological environmental protection technology, and in particular to a high temperature resistant cellulose degrading bacterial agent and a preparation method and application thereof. Background Art

[0002] Aerobic composting technology is an effective way to achieve harmless and fertilizer utilization of agricultural organic waste. Traditional composting has problems such as difficult degradation of crop straw, long maturity period, and poor product quality, which limits the development of this technology. As an important component of agricultural organic waste, crop straw usually contains a large amount of cellulose, hemicellulose and lignin. These cellulose structures are complex and poorly available to microorganisms. They are degraded very slowly during the composting process and may lead to low composting efficiency and poor composting quality. Studies have reported that the decomposition of cellulose mainly occurs during the high temperature period of composting. High temperature promotes the degradation of cellulose, accelerates the destruction of the crystallinity of straw cellulose, makes its structure loose, and accelerates the degradation rate of cellulose. Adding high temperature resistant cellulose degrading bacteria is beneficial to the increase of composting temperature, shortening the composting cycle, promoting cellulose degradation, and improving the maturity of compost products. However, high temperature resistant cellulose degrading bacteria are relatively scarce and need to be further screened in a high temperature system rich in cellulose.

[0003] A Chinese patent with publication number CN109437988A discloses a method for improving composting efficiency at low temperature by using low-temperature cellulose-degrading bacteria. The method screens low-temperature cellulose-degrading bacteria and prepares a mixed bacterial liquid to add to the combined composting of cow dung and sawdust, so as to achieve rapid heating of livestock and poultry manure composting materials under low temperature. The process focuses on screening low-temperature cellulose-degrading bacteria, without considering whether the bacteria can survive in a high-temperature composting system as the composting temperature increases. A Chinese patent with publication number CN115725447A discloses a cellulose-degrading bacterium and its application in composting. The method screens a cellulose-degrading bacterium and applies it to straw composting, thereby improving composting efficiency. The strain screened by the process is a normal-temperature cellulose-degrading bacterium, without considering whether the strain can survive in a high-temperature system, and the change in cellulose content in the composting system is not detected, and there is a lack of research on the cellulose degradation effect. The Chinese patent with publication number CN115029281A discloses a method for preparing a high-temperature resistant cellulose-degrading bacterial agent, which screens out high-temperature resistant cellulose-degrading bacteria by using a selective culture medium, and adds 2%-5% of the bacterial agent during the composting process to achieve the purpose of promoting the degradation and conversion of organic matter. This process does not consider improving the cellulase activity of the strain. The straw in the composting raw material is rich in cellulose components, and the bacteria have a weak ability to degrade cellulose. The degradation effect of cellulose in the composting system needs to be further explored. Summary of the invention

[0004] In view of this, the embodiments of the present application provide a high-temperature resistant cellulose degrading bacterial agent and a preparation method and application thereof. The high-temperature resistant cellulose degrading bacterial agent is applied to the process of sludge straw composting, and the straw can be prepared into high-quality organic fertilizer, which is of great significance for accelerating the maturity of high-temperature aerobic composting, and can effectively overcome the defects of traditional composting, such as the difficulty in degrading crop straw, long maturity cycle and poor product quality.

[0005] The first aspect of an embodiment of the present application provides a high-temperature resistant cellulose degrading bacterial agent, wherein the high-temperature resistant cellulose degrading bacterial agent is Bacillus paraglucosidase R-7; the Bacillus paraglucosidase R-7 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 1, 2024, with a deposit number of CGMCC No.30236 and a classification name of Parageobacillus thermoglucosidasius.

[0006] In some embodiments, which may include the above embodiments, the thermoglucoside-producing Bacillus R-7 is obtained by directional separation and screening in a selective medium.

[0007] In some embodiments, which may include the above embodiments, the Bacillus paragenes R-7 can grow at 55° C. and maintain organic matter degradation activity.

[0008] In some embodiments that may include the above embodiments, the high temperature resistant cellulose degrading bacterial agent is a solid bacterial agent prepared by aerobic fermentation in which the thermoglucoside paragenes Bacillus R-7 is mixed in a solid matrix.

[0009] The second aspect of the embodiment of the present application further provides a method for preparing the above-mentioned high temperature resistant cellulose degrading bacterial agent, comprising the following steps:

[0010] (1) Enrichment: Take samples of compost raw materials at the mature stage and place them in a shake flask. After shaking culture, take the suspension and inoculate it into sodium carboxymethyl cellulose liquid medium for enrichment culture to obtain enrichment solution;

[0011] (2) Spreading culture: the enriched solution is diluted with sterile water in a series of gradients to obtain dilutions of different concentrations, and the dilutions of different concentrations are pipetted into sodium carboxymethyl cellulose solid culture medium for spreading culture;

[0012] (3) Isolation and purification: Select a single colony after the coating culture and perform several streaking purification on a solid culture medium to obtain a high-temperature resistant cellulose degrading bacterial agent.

[0013] In some embodiments that may include the above embodiments, in step (1), the composting raw material is a mixture of biogas slurry and crop straw in a mass ratio of 2:1, the initial moisture content is 60-70%, the carbon-nitrogen ratio is (15-25):1, and the composting cycle is 30-40 days; or

[0014] The shaking culture conditions are: 40-60°C, 120rpm shaking culture; or

[0015] The conditions of the enrichment culture are: enrichment culture at 40-60° C. and 120 rpm shaking for 3-5 days.

[0016] In some embodiments that may include the above embodiments, in step (2), the different dilution multiples are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ;or

[0017] The coating culture specifically includes placing the culture in a 40-60° C. incubator for 3-5 days.

[0018] In some embodiments that may include the above embodiments, in step (3), the number of times of streaking purification is 5-8 times.

[0019] In some embodiments that may include the above embodiments, in step (3), the solid culture medium includes the following nutrients in mass percentage: CMC-Na 0.5%, KH2PO4 0.1%, (NH4)2SO4 0.2%, MgSO4·7H2O 0.05%, NaCl 0.05%, peptone 0.05%, yeast extract 0.05%, Congo red 0.02%, and agar 2%.

[0020] The third aspect of the embodiments of the present application also provides the use of the above-mentioned high temperature resistant cellulose degrading bacterial agent in aerobic composting of mixed biogas slurry and straw.

[0021] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0022] (1) The preparation method of the high-temperature resistant cellulose degrading bacterial agent in the present application is simple to culture, does not produce secondary pollution, and is environmentally friendly;

[0023] (2) By optimizing the enzyme production conditions using the bacterial agent obtained in the present application, the CMC enzyme activity, β-glucosidase activity and filter paper enzyme activity of Bacillus paraglucosidase were the highest when 7.5 g / L xylan was used as the carbon source, 0.15% ammonium sulfate was used as the nitrogen source, pH = 7 and the temperature was 55°C, reaching 68.25 U / mL, 60.82 U / mL and 66.89 U / mL respectively;

[0024] (3) By adding the high-temperature resistant cellulose degrading bacterial agent obtained by the present application to the compost, the temperature of the pile can reach above 50°C more quickly and last longer, which has a significant effect on improving the maturity of the compost. It is also beneficial to the degradation of cellulose and hemicellulose in the pile, thereby improving the quality of the compost. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The colony morphology and Gram staining of Bacillus paragenes in the examples of this application are shown;

[0027] Figure 2 This is a phylogenetic tree diagram of the colony of Bacillus paragenes in the examples of this application;

[0028] Figure 3 This is a graph showing the results of a filter paper strip degradation experiment of Bacillus paraproducens of thermoglucoside in the examples of this application;

[0029] Figure 4 This is a glucose standard curve diagram in the examples of this application;

[0030] Figure 5 This is a diagram showing the optimization results of the conditions for cellulose enzyme production by Bacillus paragenes in the thermoglucoside in the examples of this application;

[0031] Figure 6 This is a temperature trend diagram during the composting process of the embodiment of the present application;

[0032] Figure 7 This is a trend chart of GI during the composting process of the embodiment of the present application;

[0033] Figure 8 This is a trend chart of cellulose content during the composting process of the embodiment of the present application.

[0034] Fig. 9 This is a trend chart of the hemicellulose content during the composting process of the embodiment of the present application. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0036] Unless otherwise specified, the experimental methods used in the examples of this application are all conventional methods.

[0037] In the following examples and comparative examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0038] Example 1: Screening of thermostable cellulose-degrading bacterial agents

[0039] 1. Isolation and purification of cellulose-degrading bacteria

[0040] (1) Enrichment: Take samples of compost raw materials at the mature stage and place them in a shake flask for shaking culture (50°C, 120 rpm shaking culture). Take 5 ml of the suspension and inoculate it in 100 ml of sodium carboxymethyl cellulose liquid culture medium. Enrichment culture is carried out under the conditions of 50°C, 120 rpm shaking culture for 5 days.

[0041] (2) Spreading culture: Take 1 ml of the enrichment solution and dilute it 10 times with sterile water. The dilution multiples are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , draw 100 μL of dilution factor 10 -4 , 10 -5 , 10 -6 , 10 -7 The dilution was placed in sodium carboxymethyl cellulose solid medium and placed in a 50°C incubator for 5 days;

[0042] (3) Isolation and purification: A single colony after culture is selected and streaked on a solid culture medium (the solid culture medium includes the following nutrients in mass percentage: CMC-Na 0.5%, KH2PO4 0.1%, (NH4)2SO4 0.2%, MgSO4·7H2O 0.05%, NaCl 0.05%, peptone 0.05%, yeast extract 0.05%, Congo red 0.02%, agar 2%) for 5-8 times until a thermostable cellulose-degrading strain is obtained.

[0043] 2. Identification of thermostable cellulose-degrading bacteria:

[0044] Morphological identification of bacteria:

[0045] The purified thermostable cellulose-degrading strain was activated and inoculated on a plate using the three-zone streak method. The plate was incubated in a 50°C incubator for 2 days until a clear single colony grew on the plate. The shape, size, edge, and color of the colony were observed. Figure 1 As shown, its single colonies on the culture medium are round, wavy-edged, opaque, shiny, and slightly yellowish, and the microscopic structure is rod-shaped.

[0046] Physiological and biochemical identification:

[0047] Perform Gram staining according to the method provided by the Gram staining kit, such as Figure 1 After staining, the strain appears blue and is a Gram-positive bacterium.

[0048] Molecular biological identification of bacteria:

[0049] The total DNA of the strain was extracted, and the DNA sequence was shown in SEQ ID: NO 1. The sequencing results were obtained by PCR amplification, and 16S rRNA sequencing analysis was performed. The sequencing results were used to retrieve homologous sequences in Genbank using Blast and perform alignment analysis. Finally, a phylogenetic tree was constructed using MEGAX software, as shown in Figure 2 As shown, the strain was identified by sequence comparison as Bacillus thermoglucosidasius R-7. Bacillus thermoglucosidasius R-7 was deposited in the General Microbiology Center of China Microbiological Culture Collection Committee on April 1, 2024, with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30236 and the classification name Parageobacillus thermoglucosidasius.

[0050] Example 2: Degradation of high temperature resistant cellulose degrading bacteria filter paper strips

[0051] The thermostable cellulose-degrading bacteria were inoculated into LB medium and cultured at 50°C and 120 r / min for 24 h to prepare a bacterial suspension. 1 mL of the bacterial suspension was inoculated into a 100 mL sterile filter paper strip culture flask, and three 1×6 cm sterile filter paper strips were placed in the flask. The culture was shaken at 50°C and 120 r / min. The control group ( Figure 3 Left) No bacterial suspension was inoculated, and other conditions were the same. The degradation of the filter paper strip was observed regularly. Figure 3 As shown, the high temperature resistant cellulose degrading bacteria had a good degradation rate and efficiency for the filter paper strips. Compared with the control group, the filter paper strips could be decomposed into a paste after 5 days of culture.

[0052] Example 3: Optimization of enzyme production conditions of thermostable cellulose degrading bacteria

[0053] Optimization of enzyme production fermentation conditions of thermostable cellulose-degrading bacteria: Effects of carbon source, nitrogen source, temperature and pH on enzyme production conditions of strains during liquid fermentation.

[0054] Drawing of glucose standard curve:

[0055] Take 6 centrifuge tubes, add 0, 0.2, 0.4, 0.6, 0.8, 1.0ml of 1% glucose standard solution in turn, fill each tube with ultrapure water to 1.0ml at intervals of 0.2ml, mix with 1.0ml buffer and 2.0ml DNS reagent, boil for 5min and cool with cold water, dilute with pure water to a suitable multiple, and compare the color at a wavelength of 530nm on a UV spectrophotometer. Draw a standard curve with absorbance as the ordinate and glucose concentration as the abscissa, as shown below: Figure 4 shown.

[0056] Preparation of crude enzyme solution:

[0057] The strain was transferred to liquid enzyme-producing culture medium, cultured in a shaker at 50°C and 120 r / min for 5 days, and fresh fermentation broth was centrifuged at 8000 r / min for 10 min. The supernatant was the crude enzyme solution.

[0058] Determination of cellulase activity:

[0059] 1% CMC-Na, 0.5% salicin, 50mg filter paper were used as substrates to measure CMCase activity, β-glucosidase activity, and filter paper enzyme activity. Then, 0.5mL substrate, 0.4mL buffer and 0.1mL enzyme solution were added to the parallel test tube and the blank test tube, and the blank was replaced with the inactivated enzyme solution. After reacting in a 50℃ constant temperature water bath for 30min, 1mL DNS reagent was added and boiled in a boiling water bath for 5min. After cooling, it was diluted with pure water to an appropriate multiple and colorimetric at 530nm. The enzyme activity was calculated by the standard curve.

[0060] By optimizing the enzyme production conditions of thermostable cellulose-degrading bacteria, such as Figure 5 As shown, when Bacillus paraglucosidase used 7.5 g / L xylan as the carbon source (Figures (a) and (b)), 0.15% ammonium sulfate as the nitrogen source (Figures (c) and (d)), pH = 7 (Figure (e)) and temperature was 55°C (Figure (f)), the CMC enzyme activity, β-glucosidase activity and filter paper enzyme activity were the highest, increasing to 68.25 U / mL, 60.82 U / mL and 66.89 U / mL, respectively.

[0061] Example 4: Preparation of bacterial solution

[0062] The activated thermoglucoside-producing Bacillus was inoculated into a liquid culture medium under aseptic operation, and cultured at a constant temperature of 50° C. with shaking for 1-2 days to obtain a bacterial liquid.

[0063] Example 5: Application of thermostable cellulose-degrading bacteria in biogas slurry and straw composting

[0064] In order to study the effect of thermostable cellulose-degrading bacteria on the composting effect of biogas slurry and straw waste, two treatments were designed, with biogas slurry and straw as composting materials, with a wet weight ratio of 2:1, and the moisture content of the mixed compost material was about 60%. The bacteria-added group (Bacillus by-products of thermoglucoside) was added to the aerobic composting of straw biogas slurry at 4% of the compost mass ratio, and the control group (CK) was added with an equal amount of sterile water. The experiment lasted for 30 days.

[0065] The ambient temperature (room temperature) and the temperature change trends of the two groups are as follows Figure 6 As shown. In the initial stage of composting, the temperature of the two groups of piles was similar to the ambient temperature. Due to the low ambient temperature, the temperature rise rate of the two groups was slow. On the 4th day, the inoculated high-temperature cellulose-degrading bacteria began to play a role, and the temperature of the bacteria-added group began to be higher than that of the control group. The bacteria-added group rose to 50°C on the 10th day, entering the high temperature period one day earlier than the control group. The bacteria-added group reached a maximum temperature of 55.6°C on the 11th day and lasted for 5 days.

[0066] Seed germination index (GI) can be used as an indicator of the maturity of compost products. When GI>80%, it indicates that the compost product meets the maturity standard and has no phytotoxicity. Figure 7 As shown, at the end of composting, the GI value of the bacteria-added group (177.85%) was higher than that of the control group (171.89%), indicating that the addition of thermostable cellulose-degrading bacteria has a great effect on reducing the toxicity of compost products and improving the maturity of compost, and also improves the quality of compost products.

[0067] After activating the thermoglucoside-producing Bacillus strain, the strain was inoculated on the culture medium by the spot method and inverted in a 50°C incubator for cultivation. After vigorous growth, the strain on the solid culture medium of sodium carboxymethyl cellulose was stained with 1mg / mL Congo red solution for about 30 minutes, and then the stain was poured out and decolorized with 1mol / L NaCl solution for 30 minutes. The cellulose degradation ability of the cellulose-degrading bacteria was judged by the ratio of the transparent circle diameter to the colony diameter. Changes in cellulose content during composting are shown in Figure 2. Figure 8 As shown in the data, the cellulose content in the initial composting materials of the control group and the bacteria-added group was 19.43%. As the composting progressed, the cellulose in the pile gradually degraded and the content decreased. At the end of the composting, the cellulose contents of the control group and the bacteria-added group were 16.44% and 13.72%, respectively. The cellulose degradation rates of the control group and the bacteria-added group were 15.39% and 29.38%, respectively. It can be seen that the cellulose degradation rate of the bacteria-added group was significantly higher than that of the control group (p<0.05), which indicates that the addition of high-temperature-resistant cellulose-degrading bacteria can promote the degradation of cellulose content in the pile.

[0068] Changes in hemicellulose content during composting Fig. 9 As shown. The hemicellulose content in the initial composting materials of the control group and the bacteria-added group was 22.96%. During the composting process, the hemicellulose content of the control group decreased slightly. By the end of the 30th day of composting, the hemicellulose content dropped to 22.43%, and the degradation rate was 2.31%; while by the end of the 30th day of composting in the bacteria-added group, the hemicellulose content was 15.94%, and the degradation rate was 30.57%, which was significantly higher than that of the control group (p<0.05). This shows that the addition of high-temperature resistant cellulose-degrading bacteria is conducive to the degradation of hemicellulose content in the compost.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A high temperature resistant cellulose degrading bacterial agent, characterized in that: The thermostable cellulose degrading bacterial agent is Bacillus paragenes R-7; the Bacillus paragenes R-7 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on April 1, 2024, with a deposit number of CGMCC No.30236 and a classification name of Parageobacillus thermoglucosidasius .

2. The high temperature resistant cellulose degrading bacterial agent according to claim 1, characterized in that The high temperature resistant cellulose degrading bacterial agent is a solid bacterial agent prepared by mixing the thermoglucoside paragenes Bacillus R-7 into a solid matrix and aerobic fermentation.

3. Use of the high temperature resistant cellulose degrading bacterial agent as claimed in claim 1 or 2 in mixed aerobic composting of biogas slurry and straw.

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