Microbial agent capable of degrading lignocellulose and application of microbial agent in compost

Abstract

The invention provides a microbial agent capable of degrading lignocellulose and application of the microbial agent in compost, and belongs to the technical field of microorganisms. The microbial agent provided by the invention comprises a bacterial strain of Acidomyces acidothermophilus and a bacterial strain of Talaromyces barcinensis, and the microbial agent provided by the invention comprises a bacterial strain of Acidomyces acidothermophilus and a bacterial strain of Talaromyces barcinensis. According to the present invention, the strain Acidomyces aceticus has good growth state in the high temperature environment, and can oxidize the C-OH of hemicellulose so as to reduce the methyl and methylene structures in the lignin structure, such that the lignocellulose degradation ability is strong; the strain Talaromyces barcinensis can be used for effectively shortening the time for the compost to enter a high-temperature stage, so that the content of humus after the compost is increased. The microbial agent disclosed by the invention can effectively degrade lignocellulose in medium-temperature and high-temperature environments, and can prolong the high-temperature period of a compost body and shorten the composting time when being applied to the compost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a microbial agent capable of degrading lignocellulose and application thereof in composting. BACKGROUND

[0002] The production of agricultural products mainly consists of planting and animal husbandry, and straw is the main representative of plant residues in planting, which mainly consists of cellulose, hemicellulose and lignin, and contains a small amount of protein, amino acids and tannins, etc. At present, the main treatment methods for lignocellulose in straw are milling, grinding and rolling, pretreatment by acid or alkaline hydrolysis, physical modification, chemical modification, etc., which have high conversion rate and short time, but the reaction conditions are harsh, and when the structure of lignocellulose is destroyed, more toxic intermediate products such as furfural are formed, causing secondary pollution. Biological degradation is a green, efficient and clean environmentally friendly way to realize the conversion of straw. Using solid waste straw for composting can recycle biomass resources in nature, reduce the pressure of end-of-life disposal, make waste into treasure, and is also the basis for developing green, organic and pollution-free agriculture, which has great potential in all aspects.

[0003] Lignin is the main component of plants and is the slowest substrate to degrade, and the number of monomer units is small, but the presence of various bonds between monomer compounds (p-phenylpropane derivatives, mainly coniferyl alcohol) increases the difficulty of lignin degradation. Microorganisms mainly cut the β-1, 4 glycosidic bond and free end cellobiose through producing cellulase, and then hydrolyze to obtain glucose. Therefore, the main problem of biological degradation of lignocellulose is that the chemical composition of lignin is complex and difficult to degrade, and there is a lack of high-enzyme-activity strains. SUMMARY

[0004] Therefore, the present application aims to provide a microbial agent capable of degrading lignocellulose and application thereof in composting, which can effectively degrade lignocellulose, prolong the high-temperature period of composting and accelerate the composting process.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A microbial agent capable of effectively degrading lignocellulose, the microbial agent comprising a strain Acidomyces acidothermus and a strain Talaromyces barcinensis; wherein the strain Acidomyces acidothermus has a preservation number of CGMCC No. 22431, the strain Talaromyces barcinensis has a preservation number of CGMCC No. 22430, the preservation units are both the General Microbiological Center of the China Microbial Culture Collection Committee, and the preservation dates are both May 25, 2021.

[0007] In the composting stage, the microbial agent of the present application has a degradation rate of 60-70% for hemicellulose, 50-60% for cellulose, and 30-40% for lignin.

[0008] In the present application, the ratio of viable counts of the strain Acidomyces acidothermus and the strain Talaromyces barcinensis is preferably 1:1.

[0009] The present application also provides the use of the microbial agent described in the above technical solution in composting.

[0010] The present application also provides the use of the strain Acidomyces acidothermus in degrading lignocellulose in a high-temperature environment. The CMCase of the strain Acidomyces acidothermus in the present application can reach 1.77 U / g in a high-temperature environment.

[0011] In the present application, the high-temperature environment is preferably 50-65 DEG C. The strain Acidomyces acidothermus in the present application grows well in a high-temperature environment, can oxidize C-OH of hemicellulose, reduces the methyl and methylene structures in the lignin structure, and has strong lignocellulose degradation ability.

[0012] The present application also provides the use of the strain Acidomyces acidothermus in composting.

[0013] The present application also provides the use of the strain Talaromyces barcinensis in degrading lignocellulose in a medium-temperature environment.

[0014] In the present application, the medium-temperature environment is preferably 30-40 DEG C.

[0015] The application also provides application of the strain Talaromyces barcinensis in composting.

[0016] Beneficial technical effects: The application provides a microbial agent capable of degrading lignocellulose and application of the microbial agent in composting, and the microbial agent comprises a strain Acidomyces acidothermus and a strain Talaromyces barcinensis. The strain Acidomyces acidothermus grows well in a high-temperature environment, can oxidize C-OH of hemicellulose, reduces methyl and methylene structures in a lignin structure, and has strong lignocellulose degradation capability. The strain Talaromyces barcinensis can effectively shorten the time for composting entering a high-temperature stage, and increase humus content after composting. The microbial agent of the application can effectively degrade lignocellulose in a medium-temperature and high-temperature environment, and can prolong a high-temperature period of a composting body and shorten composting ripening time when applied in composting. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Growth curves of AA bacteria, TB bacteria+AA bacteria and TB bacteria;

[0018] Figure 2 Enzyme production curves of AA bacteria, TB bacteria+AA bacteria and TB bacteria;

[0019] Figure 3 Cellulase activities of AA bacteria, TB bacteria+AA bacteria and TB bacteria under different carbon sources;

[0020] Figure 4 Lignin peroxidase activities of AA bacteria, TB bacteria+AA bacteria and TB bacteria under different carbon sources;

[0021] Figure 5 Manganese peroxidase activities of AA bacteria, TB bacteria+AA bacteria and TB bacteria under different carbon sources;

[0022] Figure 6 Laccase activities of AA bacteria, TB bacteria+AA bacteria and TB bacteria under different carbon sources;

[0023] Figure 7 FTIR analysis of corn stalks;

[0024] Figure 8The enzyme activity of the relevant lignocellulose-degrading enzymes applied in the aerobic compost of different treatment groups; wherein (a) is CMCase, (b) is Lip, (c) is Lac, and (d) is Mnp;

[0025] Figure 9 The change of the pile temperature applied in the aerobic compost of different treatment groups;

[0026] Figure 10 The lignocellulose degradation rate in the high-temperature ending and composting stages of the aerobic compost of different treatment groups, wherein (a) is the high-temperature ending, and (b) is the composting stage. DETAILED DESCRIPTION

[0027] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited only to the following examples. The materials, reagents and the like used in the examples and test examples of the present application can be obtained from commercial channels unless otherwise specified, and the methods used in the examples and test examples of the present application are conventional methods unless otherwise specified.

[0028] The strains used in the present application have been disclosed (CN113801827A, CN114874922A) and purchased from Jiangsu College of Technology, and are also preserved in the present laboratory. For convenience, the strain Acidomyces acidothermus is simply referred to as AA bacteria, and the strain Talaromyces barcinensis is simply referred to as TB bacteria.

[0029] Example 1

[0030] 1. Materials and equipment

[0031] The raw material of the aerobic compost was the residual sludge of the sewage treatment of a food factory, and the corn straw was purchased from Zhengzhou, Henan. In the composting system as a conditioner, the ratio of the two was 60% and 40%, and a certain proportion of exogenous plant residues was added. The C / N of the pile was adjusted to 25±5, and the moisture content was 60%. After mixing, it was loaded into the compost reactor, intermittent aeration, and regular turning, and aerobic composting was carried out. The culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd. FTIR (IRTracer-100 type, Shimadzu Company) was used to observe the changes of functional groups on the surface of corn straw before and after the reaction.

[0032] The reactor for composting was a polypropylene plastic box (75 cm long x 55 cm wide x 60 cm high), and the total volume of the reactor was 248 L. A perforated sieve plate was placed at a distance of 10 cm from the bottom of the plastic box to realize uniform oxygen supply during the composting process.

[0033] 2. Experimental method

[0034] 2.1 Strain activation

[0035] A small amount of bacteria was picked from the glycerol-preserved strain freeze-dried tube at -80℃. It was inoculated into fresh nutrient LB broth medium. It was placed in a 37℃ incubator for shaking culture, and the OD 600 absorbance value of the strain was monitored until the colony grew to an appropriate density (OD 600 = 1.0).

[0036] 2.2 Cellulase activity in different carbon sources

[0037] TB bacteria, AA bacteria, and TB bacteria + AA bacteria were inoculated into 80mL-120mL LB medium containing 0.5%-2% carbon source (carboxymethyl cellulose, microcrystalline cellulose, sterilized corn straw); they were placed in a constant-temperature incubator at 37℃, 55℃, and a temperature gradient of 37℃-55℃, with a shaking speed of 150r / min-170r / min. The growth curves are shown in Figure 1 When TB bacteria + AA bacteria were mixed and cultured at a temperature gradient, the microorganisms grew the fastest. AA bacteria could survive at high temperatures of 55℃ and maintain activity.

[0038] CMCase was determined according to the national standard method, using carboxymethyl cellulose (CMC) as the substrate. 3,5-dinitrosalicylic acid (DNS) reacts with reducing sugar to produce an orange solution, and the depth of the orange color is proportional to the concentration of reducing sugar. The amount of reducing sugar was determined by colorimetry to further determine the content of CMCase. The enzyme production curve is shown in Figure 2 The optimal enzyme production of TB bacteria was 1.08U / L at 48h of culture; the optimal enzyme production of AA bacteria was 1.65U / L at 96h of culture; and the optimal enzyme production of TB bacteria + AA bacteria was 1.82U / L at 96h of culture.

[0039] The amount of cellulase produced by TB bacteria, AA bacteria, and TB bacteria + AA bacteria in the presence of 0.5%-2% different carbon sources is shown in Figure 3 Figure 3 As can be seen from the table, the CMCase of TB bacteria in LB medium with carboxymethyl cellulose, sterilized straw, and microcrystalline cellulose as carbon sources was 0.1U / mL, 0.98U / mL, and 0.5U / mL, respectively; the CMCase of AA bacteria in LB medium with carboxymethyl cellulose, sterilized straw, and microcrystalline cellulose as carbon sources was 0.26U / mL, 1.17U / mL, and 0.52U / mL, respectively; and the CMCase of TB bacteria + AA bacteria in LB medium with carboxymethyl cellulose, sterilized straw, and microcrystalline cellulose as carbon sources was 0.88U / mL, 1.73U / mL, and 0.91U / mL, respectively. When sterilized straw was added as the carbon source, TB bacteria + AA bacteria had the best performance, and the CMCase was higher than that of AA bacteria and TB bacteria.

[0040] 2.3 Ligninase activity in different carbon sources​

[0041] Inoculate TB bacteria, AA bacteria, TB bacteria + AA bacteria seed liquid in 80 mL-120 mL LB medium containing 0.5%-2% carbon source (carboxymethyl cellulose, microcrystalline cellulose, sterilized corn straw) respectively, and measure lignin-related enzyme activity.

[0042] Lignin enzymes can be mainly divided into three types: lignin peroxidase (Lip), manganese peroxidase (Mnp) and laccase (Lac). Lip mainly catalyzes the degradation of non-phenolic substrates; Mnp mainly catalyzes the degradation of phenolic and amine substrates; Lac catalyzes the oxidation of phenolic compounds and aromatic amines.

[0043] Lip determination method: accurately transfer 1-3 mL of 200 mM tartaric acid buffer into a cuvette, add 0.05 mL-0.15 mL of 40 mM veratryl alcohol, add 25 μL-35 μL of the measured fermentation liquor, and add 360 μL-380 μL of distilled water. Start the reaction by adding 0.01 mL of 20 mM H2O2 solution in a 30°C water bath, quickly measure the absorbance at 310 nm, and measure it again after 1 min. The difference between the two is the absorbance change of Lip per minute.

[0044] The results show that the lignin peroxidase of TB bacteria, AA bacteria, TB bacteria + AA bacteria in different carbon sources is as shown in Table 1. Figure 4 The Lip content of TB bacteria, AA bacteria, TB bacteria + AA bacteria in sterilized straw is the highest, which can reach 0.69 U / mL, 0.90 U / mL and 1.02 U / mL respectively.

[0045] Mnp determination method: accurately transfer 2 mL-5 mL of acetic acid-sodium acetate buffer into a cuvette, add 0.4 mL-0.8 mL of 10 mM MnSO4 solution, add 25 μL-35 μL of the measured fermentation liquor, and add 360 μL-380 μL of distilled water. Start the reaction by adding 0.06 mL of 10 mM H2O2 solution in a 30°C water bath, quickly measure the absorbance at 270 nm, and measure it again after 1 min. The difference between the two is the absorbance change of Mnp per minute.

[0046] The results show that the effect of different carbon sources on the production of manganese peroxidase by TB bacteria, AA bacteria and TB bacteria + AA bacteria is as shown in Table 2. Figure 5 The Mnp content of TB bacteria, AA bacteria and TB bacteria + AA bacteria in sterilized straw is the highest, which can reach 0.051 U / mL, 0.067 U / mL and 0.102 U / mL respectively.

[0047] Lac determination method: Accurately transfer 8 mL to 12 mL each of acetate-sodium acetate buffer and ABTS solution, mix well, incubate in a 30℃ water bath, then transfer 2 mL to 4 mL into a cuvette, zero the instrument at 420 nm, add 25 μL to 35 μL of the fermentation broth to be tested, and immediately record the absorbance value. Record the absorbance value every 30 seconds for a total of three times, take the average value, and convert it into the change in absorbance of Lac per minute.

[0048] The results showed that different carbon sources had the following effects on laccase production by TB bacteria, AA bacteria, and TB bacteria + AA bacteria: Figure 6 As shown, TB bacteria and AA bacteria had the highest Lac content in carboxymethyl cellulose, reaching 0.031 U / mL and 0.048 U / mL, respectively; AA bacteria + TB bacteria had the highest Lac content in sterilized straw, reaching 0.057 U / mL.

[0049] 2.4 FTIR analysis of corn stalks before and after degradation

[0050] Fourier transform infrared spectroscopy (FTIR) can detect changes in the chemical structure and functional groups of corn stalks before and after degradation by TB bacteria, AA bacteria, and TB bacteria + AA bacteria.

[0051] The results are as follows Figure 7 As shown, by Figure 7 It can be seen that, compared with the control group, after adding TB bacteria, AA bacteria, or TB bacteria + AA bacteria fermentation broth to corn straw for mixed culture, the lignin benzene ring structure was destroyed, the cellulose β-polysaccharide and CO bond were broken, and lignin, cellulose, and hemicellulose were partially degraded. The degradation effect of TB bacteria + AA bacteria was better than that of AA bacteria and TB bacteria.

[0052] 2.5 Actual degradation effect on lignocellulose

[0053] Sludge and straw were mixed at a ratio of (5-6):(4-5), with 10-20 kg of the mixture placed in each compost pile. Four experimental groups were set up: CK (control group), A (inoculated with AA bacteria only), B (inoculated with TB bacteria + AA bacteria), and C (inoculated with TB bacteria only). The temperature, pH, CMCase, Lip, Lac, and Mnp of the compost pile were measured. The degradation effect of lignocellulose at the end of high-temperature treatment and during the composting maturity stage was studied.

[0054] The application of single-strain and compound microbial agents in aerobic composting and the resulting changes in compost temperature, such as Figure 9 As shown. By Figure 9It can be seen that the initial temperature of each pile was around 23℃. After 1 day, the temperature of each pile rose rapidly. Group CK reached the high-temperature stage after 72 hours, with a temperature of 45℃~55℃, lasting for 1 day; Group A entered the high-temperature stage after 48 hours, with a temperature of 50℃~60℃, lasting for 3 days; Group B entered the high-temperature stage after 24 hours, with a temperature of 60℃~70℃, lasting for 5 days; and Group C entered the high-temperature stage after 24 hours, with a temperature of 50℃~60℃, lasting for 4 days. The temperature of the pile was mainly affected by the microbial biomass, which was related to the pile's moisture content and microbial activity. After inoculation with exogenous microorganisms, the microbial community structure changed, leading to changes in enzyme activity. Microbial activity was a key factor in the rapid temperature rise of the pile. Inoculation with mesophilic bacteria could shorten the temperature rise stage, allowing the pile to quickly enter the high-temperature stage, but the duration of the high temperature was short; inoculation with thermophilic bacteria could maintain bacterial activity during the high-temperature stage, prolonging the high-temperature stage of the pile. Compared with the control group, the piles inoculated with the compound bacteria all showed higher temperatures and longer durations of high-temperature periods, indicating good microbial activity, long survival time, symbiosis with indigenous microorganisms, promotion of lignocellulose degradation, and acceleration of the humification process of the pile.

[0055] The pH range of each heap was between 6 and 9. Due to the degradation of easily degradable organic matter such as proteins in the initial stage, NH3 was produced, causing the pH of each heap to rise rapidly after 1 day.

[0056] The results of CMCase, Lip, Lac, and Mnp are as follows: Figure 8 As shown, the CMCase activity in each pile reached its maximum value after the high-temperature stage, which was higher than that in the CK group (0.90 U / g). Specifically, the CMCase activity in pile B (3.96 U / g) was higher than that in pile A (1.77 U / g) and pile C (1.68 U / g), indicating that mixed inoculation can improve the cellulase activity in the pile.

[0057] In group B, Lip (1.71 U / g) was higher than in group A (1.43 U / g). Mnp activity was optimal in pile B at 1.04 U / g, significantly higher than in groups A (0.64 U / g) and C (0.61 U / g). Lip and Mnp increased sharply during the high-temperature phase. Lac content was low in all piles, with pile B showing the highest Lac content at 0.19 U / g, significantly higher than groups CK, A, and C. Pile B had high levels of CMCase, Lip, Mnp, and Lac, indicating better inoculant activity.

[0058] The effects of high-temperature treatment and the decomposition stage on the degradation of lignocellulose are as follows: Figure 10 As shown. By Figure 10It can be seen that the degradation rates of hemicellulose, cellulose and lignin in group B at the end of high temperature are 52.8%, 38.8% and 18.1% respectively, the degradation rates of hemicellulose, cellulose and lignin in group A are 33.3%, 15.4% and 8.5% respectively, the degradation rates of hemicellulose, cellulose and lignin in group C are 33.5%, 10.1% and 7.3% respectively, and the degradation rates of hemicellulose, cellulose and lignin in group CK are 20.1%, 11.0% and 1.0% respectively. The degradation rates of hemicellulose, cellulose and lignin in group B at the maturation stage are 68.2%, 51.7% and 34.3% respectively, the degradation rates of hemicellulose, cellulose and lignin in group A are 47.0%, 35.8% and 22.6% respectively, the degradation rates of hemicellulose, cellulose and lignin in group C are 40.3%, 32.7% and 20.6% respectively, and the degradation rates of hemicellulose, cellulose and lignin in group CK are 29.6%, 16.3% and 6.6% respectively. The degradation effects of lignocellulose at the end of high temperature and at the maturation stage are the highest in group B, followed by group A and group C.

[0059] After inoculation of exogenous microorganisms, the microbial community structure changes, resulting in changes in enzyme activity, and microbial activity is the key factor for rapid temperature rise of the heap. Compared with the CK group, the heaps inoculated with exogenous microorganisms all show high temperature and long duration of high temperature period, indicating that the microbial activity is good, can survive for a long time and symbiosis with indigenous microorganisms.

[0060] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, some improvements and refinements can also be made, which should be considered as the protection scope of the present application.

Claims

1. A microbial inoculum effective for degrading lignocellulose, characterized by, The microbial agent comprises a strain Acidomyces acidothermus and a strain Talaromyces barcinensis; wherein the preservation number of the strain Acidomyces acidothermus is CGMCC No. 22431, and the preservation number of the strain Talaromyces barcinensis is CGMCC No. 22430.

2. The microbial inoculant of claim 1, wherein, The ratio of viable cell numbers of the strain Acidomyces acidothermus and the strain Talaromyces barcinensis is 1:

1.

3. The microbial agent of claim 1 or 2 for use in compost.

4. The use of the strain Acidomyces acidothermus in degrading lignocellulose in a high-temperature environment.

5. Use according to claim 4, characterized in that, The high-temperature environment is 50-65 DEG C.

6. The use of the strain Acidomyces acidothermus in compost.

7. The use of the strain Talaromyces barcinensis in degrading lignocellulose in a medium-temperature environment.

8. Use according to claim 7, characterized in that, The medium-temperature environment is 30-40 DEG C.

9. The use of the strain Talaromyces barcinensis in compost.

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