CN120683022A - A Thermomylolytic Bacillus C-50-11 with High H2O2 Production and Its Application - Google Patents
A Thermomylolytic Bacillus C-50-11 with High H2O2 Production and Its ApplicationInfo
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- CN120683022A CN120683022A CN202510965635.3A CN202510965635A CN120683022A CN 120683022 A CN120683022 A CN 120683022A CN 202510965635 A CN202510965635 A CN 202510965635A CN 120683022 A CN120683022 A CN 120683022A
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Abstract
本发明属于微生物技术领域，具体涉及一株高产H2O2的嗜热淀粉芽孢杆菌及其应用。本发明中的嗜热淀粉芽孢杆菌C‑50‑11，其能够在堆肥高温期持续生成H2O2，H2O2自由基可氧化分解复杂有机物介导促进腐殖质缩合，进而有效提高了高温期堆肥中腐殖质的含量，相比于未接种堆肥中腐殖质的含量，接种后腐殖质含量提升高于20%。本发明通过结合嗜热菌的内源性H2O2强化作用，利用生物产生的氧化剂实现化学降解，充分利用了堆肥高温期的条件，为堆肥中腐殖质含量的提升提供了一种新的方法途径。
The present invention belongs to the field of microbial technology, and specifically relates to a thermophilic amyloliquefaciens strain with high H2O2 production and its application. The thermophilic amyloliquefaciens strain C-50-11 in the present invention can continuously generate H2O2 during the high temperature period of composting. The H2O2 free radical can oxidatively decompose complex organic matter to mediate and promote humus condensation, thereby effectively increasing the content of humus in the high temperature period compost. Compared with the content of humus in the uninoculated compost, the humus content after inoculation is increased by more than 20%. The present invention combines the endogenous H2O2 strengthening effect of thermophilic bacteria, utilizes biologically produced oxidants to achieve chemical degradation, makes full use of the conditions of the high temperature period of composting, and provides a new method and approach for increasing the humus content in compost.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus amyloliquefaciens C-50-11 with high H 2O2 yield and application thereof.
Background
The traditional aerobic composting has the problems of long decomposition time, large odor emission, residual rebound of Antibiotic Resistance Genes (ARGs) and the like at the high temperature of 50-65 ℃. Although the conventional microorganism strengthening means such as SBI microbial inoculum shortens the decomposition time, hydrogen peroxide which is active at high temperature is not systematically utilized to promote the mineralization of organic matters and the formation of humus.
In the current composting process, the humus formation efficiency is to be improved, and part of composting technology can not fully utilize the high-temperature period of composting to promote the effective synthesis of humus. The prior art mainly uses physical regulation and control such as ultra-high temperature fermentation or external addition of an inhibitor, and can be seen that the prior art only focuses on physical adsorption or conditioner addition, and chemical degradation is realized by no utilization of a biologically generated oxidant.
Disclosure of Invention
In order to solve the technical problems, the invention provides a bacillus amyloliquefaciens C-50-11 with high H 2O2 yield and application thereof.
The technical scheme of the invention is as follows.
The first aspect of the invention provides a bacillus stearothermophilus C-50-11 with high yield of H 2O2 accumulation, wherein the preservation number of the bacillus stearothermophilus C-50-11 is CGMCC No.33544, and the preservation date is 2025, 2 months and 17 days.
According to the invention, after kitchen waste and wood dust of a canteen of the national agricultural university (Suzhou) are mixed according to a mass ratio of 3:1 and a carbon nitrogen ratio of 25:1, water is added to enable the water content to reach 65%, then 0.25wt% of peroxymonosulfate is added for providing an active oxygen environment, composting fermentation is carried out, when the temperature of the compost is raised to be more than 50 ℃ and enters a high temperature period, a bacterium with high yield H 2O2 is screened out from the bacterium, the bacterium is named as C-50-11, and the bacterium is identified as thermophilic bacillus amyloliquefaciens. According to the bacillus amyloliquefaciens C-50-11 disclosed by the invention, H 2O2,H2O2 free radicals can be continuously generated at 50-65 ℃ to oxidize and decompose complex organic matters to mediate and promote humus condensation, so that the humus content in high-temperature compost is effectively improved, the high-temperature compost period is fully utilized, and the chemical degradation is realized by taking H 2O2 of the bacillus amyloliquefaciens C-50-11 as an oxidant, so that the decomposition speed is further accelerated.
The second aspect of the invention provides the application of the bacillus stearothermophilus C-50-11 in promoting the generation of H 2O2 in compost.
In another preferred embodiment, the Bacillus amyloliquefaciens C-50-11 is used for the production of H 2O2 during the high temperature phase of composting.
In another preferred embodiment, the high temperature period of composting refers to composting with a temperature of 50-65 ℃.
The third aspect of the invention provides an application of the bacillus amylothermophilus C-50-11 in improving the humus content in compost.
In another preferred embodiment, the increasing of humus in the compost means promoting the formation of humus in the high temperature period of the compost.
In another preferred embodiment, the high temperature period of the compost refers to the composting when the temperature reaches 50-65 ℃.
In a fourth aspect, the invention provides a method for increasing the content of humus in compost, comprising the steps of:
inoculating the bacillus amyloliquefaciens C-50-11 when the composting temperature reaches 50-65 ℃;
The inoculation amount of the bacillus amyloliquefaciens C-50-11 is 1 multiplied by 10 8CFU/g~ 1×109 CFU/g.
In another preferred embodiment, the composting fermentation is carried out by inoculating the bacillus amylothermophilus C-50-11 for 4 days to 12 days.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, kitchen waste and wood dust from a canteen of China agricultural university (Suzhou) are mixed in a mass ratio of 3:1 and a carbon nitrogen ratio of 25:1, water is added to enable the water content to reach 65%, then 0.25wt% of peroxymonosulfate is added for providing an active oxygen environment, composting fermentation is carried out, when the temperature of the compost is raised to above 50 ℃ and enters a high-temperature period, a bacterium with high yield of H 2O2 at 50-65 ℃ is screened out, the bacterium is named as C-50-11, and the bacterium is identified as bacillus amylothermophilus. H 2O2 produced by the bacillus amyloliquefaciens C-50-1 can oxidize and decompose complex organic matters, and promote humus condensation through free radical mediation. According to the invention, the bacillus amylothermophilus C-50-11 is inoculated in the high-temperature period of the compost, so that the content of humus in the compost can be effectively improved, and compared with the content of humus in the non-inoculated compost, the content of humus after inoculation is improved by more than 20%.
The bacillus amyloliquefaciens C-50-11 can improve the quality of compost products, so that the bacillus amyloliquefaciens C-50-11 is more beneficial to soil improvement and plant growth. The method has the advantages of simple operation, low cost and the like, and is easy to popularize and apply in actual composting production.
Drawings
FIG. 1 is a colony morphology of Bacillus amyloliquefaciens C-50-11 according to the present invention.
FIG. 2 is a graph showing the growth of Bacillus amyloliquefaciens C-50-11 for 48h according to the present invention.
FIG. 3 is a graph showing the results of OD 600 values of the Bacillus amyloliquefaciens C-50-11 grown for 48 hours in the present invention.
FIG. 4 is a graph showing the results of H 2O2 production by Bacillus amyloliquefaciens C-50-11 grown for 48 hours in the present invention.
FIG. 5 is a graph showing the result of growth of Bacillus amyloliquefaciens C-50-11 for 48 hours with respect to the yield of humus in compost, wherein HA represents humic acid, FA represents fulvic acid, DP represents polymerization degree, stability of humic acid structure is represented, OH is inoculated C-50-11 treatment group, and CK is control group.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
In the current composting process, the humus formation efficiency is to be improved, and part of composting technology can not fully utilize the high-temperature period of composting to promote the effective synthesis of humus. The prior art mainly uses physical regulation such as ultra-high temperature fermentation or external inhibitor, and does not combine with the endogenous H 2O2 strengthening effect of thermophilic bacteria. It can be seen that the prior art only focuses on physical adsorption or conditioner addition, and does not utilize bio-generated oxidants to achieve chemical degradation.
In the prior art, bacillus amyloliquefaciens is generally used for effectively improving methane yield and organic matter conversion rate in the anaerobic fermentation process of organic solid waste or has a proteolytic function. The invention carries out the identification of the capability of extracellular hydrogen peroxide production by 68 strains of bacteria screened from high-temperature compost, discovers that one strain of bacteria has the capability of high-yield H 2O2 at a high temperature of 60 ℃, carries out molecular biological identification, is bacillus amyloliquefaciens (Caldibacillus thermoamylovorans), is named as C-50-11, and is preserved in China general microbiological culture Collection center (CGMCC) No.33544, the preservation date is 2025, the month is 17, and the preservation address is North Star, the Barbary de 1, the national academy of sciences of China, the preservation date is 575.
The thermophilic bacillus amyloliquefaciens C-50-11 can continuously generate H 2O2,H2O2 free radicals in a high-temperature period of composting, can oxidize and decompose complex organic matters to mediate and promote humus condensation, so that the content of humus in the high-temperature period of composting is effectively improved, and compared with the content of humus in non-inoculated composting, the content of humus is improved by more than 20% after inoculation. According to the invention, through the combination of the strengthening effect of the endogenous H 2O2 of thermophilic bacteria, chemical degradation is realized by using the biologically produced oxidant, the condition of the high temperature period of the compost is fully utilized, and a new method approach is provided for improving the content of humus in the compost.
According to the conventional recognition of the invention with the publication number of CN108339828B in a kitchen waste treatment system, kitchen waste commonly called swill is food and drink produced by food and drink units (including families) and waste produced in the manufacturing process. The kitchen waste has three forms of gas, liquid and solid, namely solid dining table waste and kitchen offal, liquid oily sewage such as pot brushing water, bowl brushing water and the like, and gas-kitchen and catering lampblack.
Example 1
Isolated culture and identification of bacillus amyloliquefaciens C-50-11
(1) Isolated culture of Bacillus amyloliquefaciens C-50-11
10G of a 60 ℃ high temperature compost sample is taken and leached in 100mL of 0.85wt% NaCl to obtain leaching solution, 68 strains of bacteria which can resist the 60 ℃ high temperature are obtained by using double dilution, and the extracellular hydrogen peroxide production capacity of 68 strains of bacteria is identified, wherein one strain can produce hydrogen peroxide at 60 ℃ and is named as C-50-11. The high-temperature composting sample is obtained by mixing kitchen waste of a canteen of China university (Suzhou) and commercial wood chips in a mass ratio of 3:1 and a carbon nitrogen ratio of 25:1, adding water to ensure that the water content reaches 65%, adding 0.25wt% of peroxymonosulfate to provide an active oxygen environment, performing composting fermentation, and collecting after the temperature of the compost is raised to be above 50 ℃.
Culturing the separated C-50-11, wherein the specific process is as follows:
The culture medium comprises yeast extract 5g, tryptone 10g, sodium chloride 10g, distilled water 1000mL, pH value adjusted to 7.0,121 deg.C, and autoclaving for 20 min.
And taking single colonies to culture medium for culturing at 55 ℃ with the rotation speed of a shaking table of 160r/min for 16 hours.
(2) Molecular biological identification of strains
C-50-11 thalli are collected for bacterial whole genome sequencing, and average nucleic acid similarity (ANI) is one of the strongest measurement indexes for identifying the relatedness of bacterial genomes. The evolutionary distance of the genomic quality test is reflected based on the average of comparisons of all ortholog protein sequences of the genomic quality test, indicating that both genomes belong to the same species when ANI > 95%. The results of ANI analysis of the genome and the reference genome are shown in Table 1.
Table 1 ANI analysis results
Note that ANI represents average nucleotide identity, mapped _fragment represents mapped fragment, query_fragment represents Query fragment, taxon taxonomic group.
Example 2 detection of growth Properties of Bacillus amyloliquefaciens C-50-11
The Bacillus amyloliquefaciens C-50-11 screened in example 1 was activated in LB liquid medium and cultured at 55℃at 150pm to a logarithmic growth phase with an OD 600 of about 0.5, wherein the growth curve and OD 600 are shown in FIGS. 1 and 2.
The yield of H 2O2 was measured during the cultivation, and the results are shown in FIG. 3. It can be seen from FIG. 3 that the amount of H 2O2 was increased with time, and the amount of H 2O2 was maximized when the cultivation was performed for 40 hours.
Example 3 promotion of humus Synthesis by Bacillus amylothermophilus C-50-11
The isolated Bacillus amyloliquefaciens C-50-11 was inoculated into the medium of example 1 and cultured at 55℃and a shaking table rotation speed of 160r/min for 16 h.
Mixing kitchen waste and wood dust serving as compost raw materials, wherein the mass of the kitchen waste accounts for 75% of the mass of the compost raw materials, the mass of the wood dust accounts for 25% of the mass of the compost raw materials, when the water content in the compost raw materials is regulated to 65%, placing the mixture into a 500mL micro intelligent compost reactor for aerobic fermentation, when the temperature of the compost reaches above 50 ℃, indicating that the compost has undergone a high-temperature period, inoculating bacillus stearothermophilus C-50-11 into the compost in the high-temperature period at the moment with the inoculation amount of 10 8 CFU/g, marking the compost as an OH group, marking the compost as a CK group with the bacillus stearothermophilus C-50-11 serving as a control group, and periodically detecting the content of humus in the compost, wherein the result is shown in figure 5.
As can be seen from fig. 5, the composting of OH group was overall higher than CK group with time. When composting is carried out for 4-8 days, the content of humic acid in OH group is obviously higher than that in CK group. This indicates that Bacillus amyloliquefaciens C-50-11 promotes the formation of humic acid. The humic acid is one of main components of humic acid, can improve soil structure, increase soil fertility, and is beneficial to improving the content of humic acid in a composting product by inoculating the bacillus amylothermophilus C-50-11, thereby improving the quality of the composting product.
The fulvic acid content of the OH group was also higher than that of the CK group for most of the time. And when composting is carried out for 4-8 days, the content of the fulvic acid in the OH group is obviously higher than that of the CK group. Fulvic acid also plays an important role in soil quality and plant growth, such as regulating soil pH value, promoting nutrient absorption of plants and the like. Thus, inoculation of Bacillus amyloliquefaciens C-50-11 helps to increase the amount of fulvic acid, indicating that Bacillus amyloliquefaciens C-50-11 is able to promote the conversion of organic material during composting.
The trend of the polymerization degree DP of humic acid may reflect the stability of the humic acid structure. As can be seen from the graph, the DP value of the OH group is significantly higher than that of the CK group in most of the time, especially in 4-12 days (high temperature period of composting). This shows that inoculation of Bacillus amyloliquefaciens C-50-11 helps to increase the polymerization degree of humic acid, so that the humic acid structure is more stable, and the long-term preservation and the function in soil are more facilitated.
In addition, as can be seen from the results of FIG. 5, when the composting time is longer than 12 days, the contents of humic acid and fulvic acid are significantly reduced, and the polymerization degree of humic acid is also significantly reduced, mainly due to the rapid propagation and metabolism of Bacillus stearothermophilus C-50-11 in 4 to 12 days, and a large amount of organic substances including components such as humic acid and fulvic acid are decomposed. By day 10, metabolism may shift from the stage of rapid decomposition of organic substances to the stage of other metabolic activities using the decomposed intermediates, and at this time, a large amount of decomposed organic substances are consumed so that the precursor substances available for formation of humic acid and fulvic acid are reduced, leading to a decrease in the content of humic acid and fulvic acid, and at the same time, the decrease in the polymerization degree of humic acid may be caused by a change in the activity of enzymes or related substances involved in humic acid polymerization. Therefore, after the compost is subjected to a high-temperature period and is inoculated with the bacillus stearothermophilus C-50-11 for continuous fermentation for 10 days, the content of humus in the compost is highest, and compared with a control compost without being inoculated with the bacillus stearothermophilus, the content of humus is improved by 20%.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
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