A strain of Siamese Bacillus with lignocellulose degradation ability and its application

Abstract

The present invention discloses a strain of Siamese Bacillus with lignocellulose degradation ability and its application, which relates to the technical field of solid organic waste disposal and resource utilization. The Siamese Bacillus was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on May 27, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30775. The Siamese Bacillus provided by the present invention was isolated by the inventor from the compost of garden greening waste under different light radiation conditions. It has strong adaptability to compost with high lignocellulose content and special lighting conditions, and has strong degradation ability for lignocellulose. Applying it to compost can significantly improve the degradation efficiency of lignocellulose, increase the compost temperature, extend the high temperature period of compost, accelerate the compost maturity, and shorten the compost cycle.

Description

Technical Field

[0001] The invention relates to the technical field of solid organic waste disposal and resource utilization, in particular to a siam bacillus with lignocellulose degradation ability and application thereof. Background Art

[0002] Garden waste (GW) is a general term for natural litter (dead branches, fallen leaves, etc.) of garden plants, artificial greening prunings, and by-products of other industries (woody waste) in urban greening construction. GW has a complex structure related to the wood fiber content, about 20-30% of which is lignin, 40% is cellulose, and 20-30% is hemicellulose (Jurado et al., 2015). Disposing GW by local landfill, incineration or other environmentally harmful methods cannot achieve the goal of resource reuse (Wang et al., 2023). Composting is a relatively economical and practical method for treating garden waste. After composting, organic matter is converted into stable, hygienic compost products with excellent physical structure and nutrient content (Mistry et al., 2023). These compost products can be used as cultivation substrates or soil conditioners for plants or crops, thereby reducing the dependence on fossil fuel-based fertilizers in the cultivation of plants or crops (Mohamed et al., 2022).

[0003] GW composting is the process of converting unstable organic matter (OM) into harmless compost products through the metabolism of microbial communities under aerobic conditions (Yin and Zhang, 2021). However, the complex structure of GW is a key obstacle to its rapid decomposition during composting (Zhang and Sun, 2019). Therefore, current research on GW composting mainly focuses on accelerating the degradation of lignocellulose to improve composting efficiency and product quality. In summary, increasing the number and types of microorganisms with specific functions during the composting of landscaping waste can effectively accelerate the composting process (Hua Binbin, 2017).

[0004] Inoculation of exogenous microbial agents is the main way to increase the number of specific functional microorganisms, which can effectively improve composting efficiency and shorten the composting cycle (Cui Zongjun et al., 2002; Li Yu et al., 2008). Studies have shown that inoculation of composite microbial agents in the early stage of composting can directly increase the number and activity of functional microorganisms during the composting process and improve composting efficiency (Zhu et al., 2021). Huang et al. (2022) showed that Bacillus subtilis and Trichoderma harzianumThe inoculation increased the conversion of nutrients in the compost. Mistry et al. (2023) found that the inoculation of bacterial consortium EAc can promote the mineralization of compost. The present invention intends to develop a specific and efficient lignocellulose degrading bacterial agent and inoculate it in the composting process of garden waste, thereby effectively improving the composting efficiency of garden waste. Summary of the invention

[0005] The purpose of the present invention is to provide a Siamese Bacillus with lignocellulose degradation ability and its application, so as to solve the problems existing in the above-mentioned prior art. The Siamese Bacillus has a very strong degradation ability for lignocellulose, and its application in composting can significantly improve the lignocellulose degradation efficiency, increase the composting temperature, extend the high temperature period of composting, accelerate the maturity of composting, and shorten the composting cycle.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a siam bacillus having lignocellulose degradation ability ( Bacillus siamensis ), the Siamese Bacillus was deposited in the General Microbiology Center of China Culture Collection Administration on May 27, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30775.

[0008] The present invention also provides the use of the above-mentioned Bacillus siamensis in preparing a microbial agent for composting.

[0009] The present invention also provides a microbial agent for composting, comprising the above-mentioned Bacillus siamensis.

[0010] The present invention also provides a method for preparing the above-mentioned microbial agent, comprising the steps of culturing the above-mentioned Siamese Bacillus using a culture medium, separating the bacterial bodies, and then resuspending the bacterial bodies with physiological saline to obtain the microbial agent.

[0011] The present invention also provides application of the above-mentioned siam bacillus or microbial agent in composting.

[0012] Furthermore, the raw material of the compost is landscaping waste.

[0013] Furthermore, the composting adopts a lighting condition without blue light radiation.

[0014] The present invention also provides a method for improving composting efficiency, which is characterized by comprising the steps of crushing the compost raw materials, adding the above-mentioned microbial agent, and performing composting.

[0015] Furthermore, the added amount of the microbial agent is 0.5wt%.

[0016] Furthermore, the composting adopts a lighting condition without blue light radiation.

[0017] The present invention discloses the following technical effects:

[0018] (1) The Siamese Bacillus provided by the present invention is isolated by the inventors from the compost of landscaping waste under different light radiation conditions, and has strong adaptability to compost with high lignocellulose content and special light conditions.

[0019] (2) The microbial agent prepared by the present invention can efficiently secrete enzymes such as manganese peroxidase, laccase, cellulase and lignin peroxidase, and has a strong ability to degrade lignocellulose. Adding the microbial agent prepared by the present invention during the composting process can significantly improve the efficiency of lignocellulose degradation, increase the composting temperature, extend the high temperature period of the composting, accelerate the maturity of the composting, and shorten the composting cycle. At the same time, the microbial Alpha diversity in the compost product prepared by adding the microbial agent prepared by the present invention is improved, and the succession of the microbial community structure in the compost is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a statistical chart of temperature changes during the composting process of each treatment group;

[0022] Figure 2 It is the statistical diagram of pH change during composting of each treatment group;

[0023] Figure 3 It is the statistical diagram of conductivity change during composting process of each treatment group;

[0024] Figure 4 It is a statistical chart of the changes in organic matter content during the composting process of each treatment group;

[0025] Figure 5 It is a statistical chart of the changes in lignin content during the composting process of each treatment group;

[0026] Figure 6 It is a statistical chart of the changes in cellulose content during the composting process of each treatment group;

[0027] Figure 7Statistical chart of changes in hemicellulose content during composting in each treatment group. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The formula of the culture medium used in the following examples is as follows:

[0034] Beef extract peptone medium: beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L and agar 20g / L.

[0035] Potato dextrose agar medium (PDA): potato powder 6g / L, agar 20g / L and glucose 20g / L.

[0036] Sodium carboxymethyl cellulose medium: sodium carboxymethyl cellulose 10g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate heptahydrate 0.1g / L, ferrous sulfate heptahydrate 0.1g / L, manganese sulfate 5×10-4g / L, peptone 10g / L, yeast extract powder 10g / L and agar powder 20g / L.

[0037] Aniline blue medium: When culturing fungi, add 0.1g of aniline blue to each liter of PDA medium; when culturing bacteria, add 0.1g of aniline blue to each liter of beef extract peptone medium.

[0038] Guaiacol medium: When culturing fungi, add 0.4 mL of guaiacol to each liter of PDA medium; when culturing bacteria, add 0.4 mL of guaiacol to each liter of beef extract peptone medium.

[0039] Example 1 Isolation, identification and preservation of lignocellulose-degrading bacteria

[0040] 1. Isolation of lignocellulose-degrading bacteria

[0041] Take samples from the heating, high temperature, cooling and maturity stages of the garden waste compost, weigh 10g of the sample and put it into a sterilized 100mL plastic bottle (sterilized at 121℃ for 20min, this sterilization condition is applicable to the sterilization of all materials), add 90mL of sterile water and place the plastic bottle on a reciprocating shaker for 15min to allow the microorganisms to fully disperse into the liquid phase, and let it stand for 15min. Pipette 0.1mL of the supernatant into a 2mL sterile centrifuge tube containing 0.9mL of sterile water, shake it thoroughly, and obtain 10 -1 Dilution, according to the above method to prepare 10 -2 Up to 10 -6 Dilution: 0.1 mL of dilution was applied to beef extract peptone medium and potato dextrose agar medium to culture bacteria and fungi, respectively. The medium was placed in a constant temperature incubator (record the temperature) and cultured until clear colonies grew. Colonies with different morphologies were picked and placed in new culture medium, and streaked and cultured under the same conditions until single colonies or colonies with relatively uniform bacterial morphology appeared, completing the strain purification.

[0042] Then identify the lignin and cellulose degradation ability of the microorganism to complete the primary screening. Prepare several filter paper pieces with a diameter of 6 mm, sterilize them and neatly place them on sodium carboxymethyl cellulose medium, aniline blue medium, and guaiacol medium with tweezers to identify the strain's cellulase production ability (sodium carboxymethyl cellulose medium), manganese peroxidase or lignin peroxidase production ability (aniline blue medium, both manganese peroxidase and lignin peroxidase can fade aniline blue), and laccase production ability (guaiacol medium). The bacteria preserved in the slant medium were propagated, and the sterilized bamboo sticks were used to dip each culture to wet the filter paper to complete the inoculation. This method can concentrate the culture within the filter paper to form a regular fading circle or color change circle for observation. Isolate microorganisms that can produce any of the above phenomena to complete the primary screening; identify the antagonistic effects of the selected microorganisms to complete the rescreening. Finally, a strain was screened out and named B2.

[0043] 2. Strain Identification

[0044] Morphological, physiological, biochemical and molecular biological identification of strain B2:

[0045] Morphological identification: The colonies are round, white, dry and wrinkled; the colonies are yellow and opaque, with a rough surface, ridges, irregular edges, and a biofilm when the liquid culture is static; Gram staining is positive, rod-shaped, and forms endospores that are oval.

[0046] Physiological and biochemical identification: starch and gelatin hydrolysis, acetylmethylcarbinol (VP) test was negative, nitrate reduction test was negative, phenylalanine deaminase test, indole test, methyl red (MR) test, hydrogen sulfide test were all negative.

[0047] Molecular Biology Identification:

[0048] The strain B2 was identified by 16S rDNA. The 16S rDNA sequence (SEQ ID NO.1) obtained by sequencing was compared with the reported sequences in the NCBI GenBank database by BLAST. The results showed that B2 was similar to Bacillus siameensis ( Bacillus siamensis ) have a homology of more than 99%.

[0049] Combining the morphological characteristics, physiological and biochemical test results and molecular biological identification results, B2 was identified as Bacillus siamensis ( Bacillus siamensis ).

[0050] SEQ ID NO.1:

[0051] GGGGGTTACGTCATCCCCACCTTCCTCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACC TGGTAAGGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCAAATCCTGTTAGTCCAC.

[0052] 3. Strain preservation

[0053] Bacillus siame B2 was deposited at the General Microbiology Center of China National Culture Collection Administration on May 27, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30775.

[0054] Example 2 Preparation of microbial agent

[0055] On a sterile operating table, inoculate 5% of the seed liquid of strain B2 into LB liquid culture medium, and culture at 200r / min, 37°C, with constant temperature shaking for 24h. When the free bacteria are in the logarithmic growth phase, take them out, centrifuge them at 4000r / min for 20min at room temperature, discard the supernatant, wash the wet bacterial surface several times with sterilized 0.9% saline, adjust it into a bacterial suspension with saline, and make the absorbance of the bacterial suspension reach 1.003 at a wavelength λ of 600nm to obtain the microbial agent, which is stored for later use.

[0056] Example 3 Application of liquid bacterial agent in composting of landscaping waste under specific light wavelength conditions

[0057] 1. Production of landscaping waste compost

[0058] The landscaping waste was composed of residual branches and leaves collected during spring landscape maintenance. Before composting, the collected landscaping waste was mechanically crushed into pieces <2 cm long to obtain a raw material mixture. The experiments were conducted in a composting plant, and all the initial total amounts (total dry weight = 80 kg) were the same. The treatment without the addition of microbial inoculants (control) was designated as T3. The treatments containing 0.5wt% microbial inoculants (Example 2) and 0.5wt% common commercial inoculants (EM inoculants) were designated as T1 and T2, respectively.

[0059] Each treatment group composted in the following manner: the raw material mixture was piled under a light-transmitting film device with a bottom area of ​​1m 2 , a cone 1 m high. After the pile is built, 8 beech wood sticks are used to build a cubic wooden frame with a side length of 1.5 m, placed outside the pile, and a light-transmitting film is laid on the wooden frame, and small holes are evenly punched at intervals of 10 cm on the surface of the light-transmitting film to ensure ventilation. During the composting process, the pile is turned over every 3 days to ensure gas exchange in the pile. At the same time, the pile is properly replenished with water to keep the moisture content of the pile at around 60%. During the composting process, a thermometer is used to measure the temperature at three different locations in the middle of the pile every day, and the average value is calculated. To avoid systematic errors, the same thermometer is used to measure the ambient temperature. When the temperature of a certain pile is equal to or lower than the ambient temperature, it means that the composting cycle is completed. When the temperature of all piles is equal to or lower than the ambient temperature, it means that the composting cycle of all treatments is completed.

[0060] Sampling was carried out on days 0, 3, 7, 11, 16, 20, 24, 28, 32, 36 and 39 of composting. The collected samples were divided into two parts: one part was stored at 4°C for fresh sample analysis and microbial diversity measurement; the other part was air-dried and used for the analysis of physicochemical properties.

[0061] 2. Experimental results

[0062] 2.1 Temperature changes during composting

[0063] Taking the treatment without adding bacterial agent (T3) as the control, the composting experiment of landscaping waste was carried out under the condition of no blue light radiation. The temperature change trend of the pile in all treatment groups was basically the same, that is, the temperature rose rapidly into the thermophilic period and reached the peak, and then slowly decreased ( Figure 1). T1 reached 50.6℃ on the 4th day of composting and entered the thermophilic period earlier than other treatments. The peak composting temperature and occurrence time of each treatment are as follows: T1 (65.8℃, 8th day), T2 (61.2℃, 10th day), T3 (58.4℃, 8th day). The thermophilic periods of T1-T3 were 12, 9 and 6 days, respectively. It is generally believed that the thermophilic period of composting lasts for more than three days, and harmful substances such as pathogenic microorganisms are eliminated. Therefore, all treatments meet the requirements of "harmless" composting treatment. The composting temperatures of T1-T3 returned to ambient temperature after 35, 37 and 39 days, respectively.

[0064] 2.2 Changes in pH during composting

[0065] Taking the treatment without adding microbial agent (T3) as the control, the composting experiment of landscaping waste was carried out under the lighting conditions without blue light radiation. The changes of pH values ​​in each treatment were similar, that is, the trend of first increasing and then decreasing. When the pH value of compost is between 6.5 and 7.5, its product is generally considered to be stable. In this experiment, only the pH value of the compost product of T3 (without adding microbial agent) was not uniformly within the above range. The pH value of the compost product of T3 was 7.62, which was higher than that of T1 and T2 ( Figure 2 ).

[0066] 2.3 Changes in conductivity during composting

[0067] In addition, during the composting process, the conductivity showed a trend of first rapidly increasing to a peak value and then slowly decreasing. Figure 3 As shown in the figure, the compost product of T1 has the highest conductivity value, and the compost product of T3 (without added microbial agent) has the lowest conductivity value. Under the same lighting conditions (no blue light radiation), the reason why the conductivity value of T2 is lower than that of T1 may be that more organic matter and lignocellulose in T1 are converted into small-molecule inorganic nutrients, resulting in a higher overall nutrient content in the compost. The reason why the EC value of T3 is lower may be that the lower microbial activity leads to weaker mineralization of organic matter and less inorganic nutrients produced.

[0068] 2.4 Changes in organic matter during composting

[0069] Taking the treatment without adding bacterial agent (T3) as the control, the composting experiment of landscaping waste was carried out under the lighting condition without blue light radiation. The organic matter content in all treatments showed a downward trend ( Figure 4). In the first 3 days of composting, the degradation rate of organic matter was very slow, but the fastest degradation rate was observed on the 7th to 11th day of composting. At the end of composting, the organic matter degradation rates of T1-T3 were 60.6%, 56.7% and 49.1%, respectively. The organic matter content of the compost products of T1-T3 was all <600 g / kg, and the products could be considered stable and mature. Under the same light conditions and composting environment, the highest final organic matter degradation rate during composting was in T1, followed by T2, and T3 had the worst organic matter degradation effect.

[0070] 2.5 Changes in lignocellulose during composting

[0071] Taking the treatment without adding bacterial agent (T3) as the control, the composting experiment of landscaping waste was carried out under the condition of no blue light radiation. The contents of lignin, cellulose and hemicellulose showed a decreasing trend during the composting process ( Figure 5-7 ). Similar to the degradation rate of lignin, the degradation rates of cellulose and hemicellulose were fastest during the thermophilic period and then gradually slowed down. Figure 5 It was found that the lignin degradation rate was the highest in T1 and the lowest in T3. The lignin degradation rates of each treatment were arranged in descending order as follows: T1 (43.2%) > T2 (36.0%) > T3 (31.4%). Figure 6 As shown in the figure, the cellulose degradation rates are as follows: T1 (51.0%) > T2 (46.6%) > T3 (37.9%). Figure 7 As shown in the figure, the degradation rate of hemicellulose is from high to low: T1 (44.5%) > T2 (40.6%) > T3 (37.6%). The degradation rate of lignocellulose treated with T2, which was added with common commercial microbial agents containing multiple microorganisms, was lower than that of T1, which was only added with dominant degradation agents. This may be attributed to the ability of the homemade microbial agent itself to efficiently produce lignocellulose degrading enzymes (including lignin peroxidase, manganese peroxidase, laccase and cellulase), and has a specific degradation function for the main components of landscaping waste compost (lignin, cellulose and hemicellulose). At the same time, since Siamese Bacillus B2 is an original microorganism screened from landscaping waste compost, it has a strong adaptability to the composting environment, avoiding the disadvantage of poor effect caused by the inability of exogenous microorganisms to adapt to the landscaping waste composting environment.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus siamensis with the ability to degrade lignocellulose ( Bacillus siamensis ), characterized in that, The Siamese Bacillus was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 27, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30775.

2. Use of the siam bacillus as claimed in claim 1 in preparing a microbial agent for composting.

3. A microbial agent for composting, characterized in that: The invention comprises the siam bacillus as described in claim 1.

4. A method for preparing the microbial agent according to claim 3, characterized in that: The method comprises the steps of culturing the siam bacillus according to claim 1 by using a culture medium, isolating the bacterial body, and then resuspending the bacterial body with physiological saline to obtain the microbial agent.

5. Use of the siam bacillus according to claim 1 or the microbial agent according to claim 3 in composting.

6. The use according to claim 5, characterized in that: The raw material of the compost is gardening waste.

7. The use according to claim 5, characterized in that: The composting adopts a lighting condition without blue light radiation.

8. A method for improving composting efficiency, characterized in that: The method comprises the steps of crushing the compost raw materials, adding the microbial agent according to claim 3, and performing composting.

9. The method according to claim 8, characterized in that The added amount of the microbial agent is 0.5wt%.

10. The method according to claim 8, characterized in that The composting adopts a lighting condition without blue light radiation.

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