CN114410520B - Bacillus subtilis and application thereof in fecal compost deodorization - Google Patents
Bacillus subtilis and application thereof in fecal compost deodorization Download PDFInfo
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- CN114410520B CN114410520B CN202210026403.8A CN202210026403A CN114410520B CN 114410520 B CN114410520 B CN 114410520B CN 202210026403 A CN202210026403 A CN 202210026403A CN 114410520 B CN114410520 B CN 114410520B
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- bacillus subtilis
- ybs01
- compost
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- deodorization
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Classifications
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/80—Separation, elimination or disposal of harmful substances during the treatment
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel bacillus subtilis (Bacillus subtilis) and application thereof in fecal compost deodorization. The bacillus subtilis sieve is selected from the waste of the Kadsura et al, has a fixing effect on organic nitrogen and sulfur elements in compost raw materials, can effectively reduce the release of odor such as ammonia gas, hydrogen sulfide and the like, accelerates the manure composting process, improves the quality of organic fertilizers, and is preserved in China center for type culture collection of Wuhan university in China in 1 month 13 of 2021, wherein the preservation number is CCTCC NO: m2021053.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening, in particular to novel bacillus subtilis and application thereof in deodorization of fecal compost.
Background
Along with the continuous improvement of the living standard of people, the demand for livestock and poultry products is continuously increased, and according to the data of the United nations grain and agriculture organization (FAO), the demand for meat is increased by 73% all over the world by 2050, so that the demand for meat is met, the livestock industry needs to rapidly develop from the traditional free-range cultivation mode to the intensive and large-scale direction, and accordingly, a large amount of produced livestock and poultry manure becomes an important environmental pollution source. The stacking of a large amount of livestock and poultry manure occupies the land, and the produced harmful gas pollutes the environment and seriously endangers human health, and affects the exertion of the potential production performance of livestock and poultry, so that the harmless and recycling treatment of the livestock and poultry manure is enhanced, and the method is an urgent need for realizing ecological environment protection and animal husbandry development.
The livestock manure contains protein, carbohydrate and the like, the protein is decomposed into nitrate substances under the aerobic condition, malodorous gases such as ammonia, hydrogen sulfide, methylamine and the like are decomposed under the anaerobic condition, and the ammonia concentration discharged into the air is too high to form acid sedimentation, so that water and soil are polluted. The decomposition of carbohydrates into greenhouse gases such as methane and carbon dioxide causes global warming, and about 50% of the global warming contribution comes from the aquaculture industry. The odor generated by the livestock and poultry manure endangers the health of the farmers and surrounding residents. Mainly harms the nervous system, the respiratory system, the circulatory system, the digestive system, the endocrine system and other harms, such as ammonia and aldehydes can stimulate the eyes to have symptoms of lacrimation, conjunctivitis, corneal edema and the like.
At present, the domestic and foreign animal manure malodor treatment mainly comprises physical, chemical and biological deodorization technologies, and the physical, chemical and biological effects are utilized to change the phase and structure of malodorous substances so as to achieve the deodorizing effect. The physical deodorization technology is to use an adsorbent and a masking agent to temporarily adsorb and mask odor to achieve the deodorization effect, but the deodorization purpose is not achieved fundamentally. The chemical deodorization technology is to react the added chemical substances with malodorous substances to achieve the purpose of deodorization, and comprises an oxidation method and a catalytic oxidation method. The oxidation method is a process in which a strong oxidizing agent oxidizes a reducing malodorous substance into a harmless odorless substance. Ozone and ferrate are currently commonly used as oxidants. The catalytic oxidation method is to oxidize organic malodorous substances under the action of a catalyst to achieve the purpose of deodorization, such as hydrocarbon, aldehyde and alcohol organic substances. The chemical deodorization technology has good deodorization effect, but has the defects of high cost, complex reaction, secondary pollution and the like, and is not easy to popularize and use. The biological deodorization technology is a technology developed in the 50 s of the last century, and research is conducted in the field in many countries of the 70 s, wherein japan, germany and the like have achieved remarkable results in terms of deodorization methods and basic principles, deodorization devices and operation processes, deodorization microorganisms, deodorization kinetics and the like, and the research work in this aspect of China is later, and the measurement and research work of malodorous gas are conducted until the end of the 80 s. The microbial deodorizing technology utilizes the metabolism of microbe to degrade malodorous components into odorless and harmless end products, such as H 2O、CO2, etc. to reach the deodorizing aim. The microbial deodorization technology has obvious deodorization effect on odor and low requirement on environmental conditions, and the energy required by growth and propagation is oxidation energy generated in the oxidative decomposition process, so that other nutrient substances are not needed, and the running cost is low.
For example, feng Jian is separated and screened to obtain deodorizing strain with good deodorizing effect on animal feces. Chen Liyuan and the like separate strains 10MG from soil around a farm, so that the release amount of fresh pig manure NH 3 and H 2 S can be reduced by 67.95 percent and 26.6 percent, and Liu Chunmei and the like study the influence of the screened high-temperature resistant deodorizing strains on the release amount of NH 3 and H 2 S and the conversion of substances, and the results show that the deodorizing strains can effectively reduce the release of NH 3 and H 2 S and retain nutrients such as nitrogen, sulfur and the like. However, due to the complex odor components, the deodorizing effect of a single microorganism is limited, and the deodorizing effect of the combination of a plurality of microorganisms is stronger. She Fenxia and the like are mixed and cultured to prepare the compound microbial deodorant, so that the concentration of NH 3 and H 2 S in a pig house can be reduced by 78.4 percent and 66.7 percent respectively, and the concentration of NH 3 and H 2 S in a pig manure composting field can be reduced by 84.4 percent and 62.1 percent respectively. Gao Ying and other researches on the deodorizing effect of different types of microorganisms on pig manure show that lactobacillus acidophilus and bacillus pumilus have good NH 3 removing effect and streptomyces microflavus, rhizopus oryzae and saccharomyces boulardii have good H 2 S removing effect.
Functional microorganisms play a key role in the biological deodorization process of the livestock manure, so that the key of biological deodorization is to screen high-efficiency deodorization microorganisms. For a long time, attempts have been made to improve new methods and approaches for optimizing the screening of deodorizing microorganisms, and the deodorizing effect of the deodorizing microorganisms screened is not ideal due to limitations of the screening methods, specificity of the microorganisms, and the like, so that the screening of deodorizing microorganisms with high efficiency is of great significance.
Disclosure of Invention
The invention provides a novel bacillus subtilis (Bacillus subtilis) and application thereof in deodorization of manure compost for solving the problems in the prior art. The bacillus subtilis sieve is selected from waste of raising of Kaempferia galanga chickens in Kaempferia galanga, has a fixing effect on organic nitrogen and sulfur elements in compost raw materials, can effectively reduce release of ammonia and hydrogen sulfide, improves quality of organic fertilizers, and has wide application prospect.
In one aspect, the invention provides bacillus subtilis YBS01 (Bacillus subtilis YBS 01) which has been preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China on 1 month 13 of 2021: m2021053.
In one aspect, the invention provides application of the bacillus subtilis in biological deodorization.
In one aspect, the invention provides application of the bacillus subtilis in compost production.
The invention also provides a biological deodorant which comprises the bacillus subtilis YBS01.
The biological deodorant also comprises any one or a combination of two or more of bacillus, aspergillus, saccharomycetes and lactobacillus.
The living bacterial amount of the bacillus subtilis YBS01 in the biological deodorant is at least 10 8 CFU/g.
The invention also provides application of the biological deodorant in deodorization of fecal compost.
The feces is livestock and poultry feces.
The feces are chicken manure, duck manure, pig manure, cow manure and sheep manure.
Advantageous effects
The bacillus subtilis YBS01 obtained by screening can produce protease, amylase and cellulase in high yield, wherein the enzyme activity of the protease in the fermentation supernatant of the bacillus subtilis is 26.6U/ml.
The bacillus subtilis YBS01 can be independently applied to manure composting, can obviously reduce the emission of odor gases such as ammonia gas, hydrogen sulfide and the like, can obviously improve the composting temperature, accelerates the manure decomposition process, and improves the decomposition degree and fertilizer efficiency. Wherein:
(1) The peak value of odor release is reduced by 56% -58%, the release concentration of ammonia gas is reduced by 65% at the highest, the release concentration of hydrogen sulfide is reduced by 60% at the highest, and the chicken manure of the treatment group 2 and the treatment group 3 completely eliminates the discharge of hydrogen sulfide on the 3 rd day; the highest temperature of the chicken manure piles of the treatment group reaches 64-68 ℃, the average temperature reaches 51-55 ℃ which is obviously higher than that of the control group (55 ℃), the decomposition degree is obviously higher than that of the control group (45 ℃), and the decomposition degree is improved by 170.7% and is up to 76.21% compared with that of the control group;
(2) The peak value of ammonia release is reduced by 79% -88% and the release concentration of ammonia is reduced by 88% at most when the bacillus subtilis YBS01 bacterial powder is inoculated to the treated cow dung; the instantaneous release amount of the hydrogen sulfide in the cow dung of the treatment group is reduced to 0 in the 3 rd to 4 th days of fermentation, and is 2 to 3 days earlier than that of the control group; the highest temperature of the cow dung pile body of the treatment group reaches 66-69 ℃, is obviously higher than that of a control group (65 ℃), the average temperature reaches 54-56 ℃, and is also obviously higher than that of the control group (42 ℃), so that the decomposition degree is improved by 64 percent and is up to 80 percent compared with that of the control group;
(3) The peak value of ammonia release is reduced by 55% -71% and the maximum ammonia release concentration is reduced by 71% when the bacillus subtilis YBS01 bacterial powder is inoculated to the pig manure; the peak value of the release of the hydrogen sulfide is reduced by 76% -80%, and the release concentration of the hydrogen sulfide is reduced by 97% at most; the pig manure of the treatment group completely eliminates the emission of hydrogen sulfide on the 5 th to 6 th days of fermentation, and is remarkably earlier than that of the control group. The highest temperature of the pig manure pile body of the treatment group reaches 66-69 ℃, which is obviously higher than that of the control group (60 ℃), the average temperature reaches 56-57 ℃, which is also obviously higher than that of the control group (46 ℃), and the decomposition degree is increased by 130% and up to 78% compared with that of the control group.
The bacillus subtilis YBS01 can be also compounded with a decomposing inoculant to be applied to manure composting, and the decomposing effect is better. Compared with a control group added with the decomposed inoculant alone, the decomposition degree of chicken manure of a treatment group added with the bacillus subtilis YBS01 bacterial powder is obviously improved, and the decomposition degree is further increased by up to 121.23 percent along with the increase of the addition amount of the bacillus subtilis YBS01, and is improved by 52.2 percent compared with the control group. In addition, the bacillus subtilis YBS01 can also obviously improve the composting temperature of chicken manure, and compared with a control group, the highest composting temperature and the average composting temperature of the treatment group are respectively improved by 8 ℃ and 4 ℃, the decomposing period of 3 days is shortened, and unexpected technical effects are achieved.
The bacillus subtilis YBS01 provided by the invention can be used as a deodorizing bacterial agent, an organic fertilizer composting inoculant and the like, can be widely applied to the field of agricultural production, and has remarkable effect and wide application prospect.
Drawings
FIG. 1 is a plate colony morphology of YBS01 strain;
FIG. 2 shows a MALDI-TOF MS mass spectrum of YBS01 strain.
Detailed Description
The equipment and reagents used in the embodiments of the present invention may be selected from any of those commercially available. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention.
The medium composition used in the examples:
enrichment culture medium ：MgCl2·6H2O 0.8g,NH4Cl 1.0g,KH2PO4 2.0g,NaHCO3 2.0g,Na2S2O35.0g, distilled water 1000mL, pH 6.8-7.2, sterilizing at 121 ℃ for 20min;
isolation medium: adding 15-20g of agar powder into the liquid enrichment culture medium to obtain a solid culture medium.
The rotten inoculant used in the examples was a rotten fermented inoculant source kangbao, 50 hundred million cfu/g purchased from Shandong blue biotechnology Co.
The invention is further illustrated below in connection with specific examples.
EXAMPLE 1 deodorant Strain screening
1. Sample of
Collecting waste from the cultivation of the peggarete chickens.
2. Enrichment culture and purification and separation of strains
1G of chicken manure is added into a culture flask containing 99mL of sterile water, a plurality of glass beads are added to prepare a suspension, then 1mL of the suspension is taken and added into a culture flask containing 50mL of enrichment medium, the sealing is carried out, and the culture flask is placed in a biochemical incubator at 30 ℃ for static culture for 3-7d. The enrichment culture has more gas production and high turbidity, is coated on a separation culture medium, is cultured for 2 days at 30 ℃, and is respectively picked out to form single colonies, and is continuously purified for 2-3 times on a nutrient agar culture medium. The purified single colonies were inoculated into 50mL of LB liquid medium, respectively, and cultured at 37℃for 14h at 220 r/min. The number of effective viable bacteria in the fermentation broth of each strain is detected by adopting a plate counting method.
The applicant respectively names 3 strains with highest effective viable bacteria numbers as YBS01, YBS02 and YBS03, and the effective viable bacteria numbers in fermentation liquor are respectively 2.76 multiplied by 10 8CFU/ml,2.12×108CFU/ml,2.83×108 CFU/ml.
2. Compound screen for ammonia and hydrogen sulfide degradation strain
(1) Effect of reducing nitrogen
Three strains YBS01, YBS02 and YBS03 obtained by primary screening are respectively inoculated into LB liquid culture medium, cultured for 24 hours at 30 ℃ and 150r/min, centrifugally collected, and resuspended in normal saline to prepare bacterial suspension with the concentration of 0.5 multiplied by 10 2 CFU. The bacterial suspension was inoculated into a nitrogen reduction screening medium (NaNO 2 1g,CH3 COONa 4.8g, agar 15g, water was added to 1L, pH 7.5) at a volume ratio of 1%, and cultured at 30℃for 20 hours under 150 r/min.
After the culture is finished, sampling and measuring OD 600, 8000r/min, centrifuging for 10min, taking supernatant to measure nitrite nitrogen concentration, and calculating nitrite nitrogen and total nitrogen removal rate. The specific results are shown in Table 1.
TABLE 1 degradation Effect of the Primary screening strains on nitrite Nitrogen and Total Nitrogen
| Strain | Nitrite nitrogen removal rate | Total nitrogen removal rate |
| YBS01 | 70％ | 65％ |
| YBS02 | 55％ | 47％ |
| YBS03 | 64％ | 51％ |
From the results in Table 1, the YBS01 strain in the three strains obtained by the primary screening has the strongest degradation capability on nitrite nitrogen and total nitrogen, the removal rate is respectively up to 70% and 65%, and the effect is very remarkable.
(2) Sulfur reducing effect
Respectively inoculating three strains YBS01, YBS02 and YBS03 obtained by primary screening into an LB liquid culture medium, culturing for 3d at 30 ℃ and 150r/min to obtain seed liquid, and adjusting the concentration of the seed liquid to about 10 6 CFU/ml;
100ml of a liquid selection medium (peptone 10g, beef extract 2g, naCl 2 5g,Na2S.9H2O2 g, distilled water 1L) for the desulphurisation bacteria was added to a 250ml triangular flask, the seed solution was inoculated, and the culture medium without the inoculation bacteria solution was used as a blank control, and the culture was allowed to stand at 30 ℃. The concentration of sulfide in the medium was periodically determined by iodometry. At the same time, the percentage of sulphide content in the inoculated medium was calculated as 100% of the sulphide concentration in the blank medium.
Percent sulfide (%) = sulfide concentration in inoculation medium/sulfide concentration in blank medium x 100%.
The results show that the sulfide content in the culture medium is reduced to 27% of the blank control group on the 2 nd day after the YBS01 strain is inoculated; by day 3, the content of sulfide in the culture medium can not be detected, the sulfur reduction effect is obvious, and unexpected technical effects are achieved. But the sulfur removal effect of YBS02 and YBS03 strains is not obvious, and sulfide is still detected on the 4 th day of culture.
The results show that the YBS01 strain has the strongest degradation capability on nitrite nitrogen and sulfide in three strains obtained by screening.
EXAMPLE 2 identification of YBS01 Strain
2.1 Colony morphology identification
The bacterial colony of YBS01 strain is shown in figure 1, the bacterial colony is light yellow, the diameter is 5-57mm, the edge of the bacterial colony is rough and opaque, the middle is flat, the bacterial body is short straight rod-shaped, and the bacterial colony can generate spores. The spore is elliptical, the part of the spore is circular, and the sporangium is not inflated. Cells are present singly, in pairs or in short chains.
2.2 16S rDNA molecular identification
And extracting the genome of the YBS01 strain by using the kit. Then, the genome is used as a template, and the 16S rDNA is amplified by using a specific primer. The amplified PCR products were subjected to 1% agarose gel electrophoresis and were sent to sequencing company for sequencing.
Sequencing results show that the sequence of the PCR amplification product is SEQ ID NO. 1. The sequence was found to have the highest similarity to Bacillus subtilis (Bacillus subtilis) by BLAST alignment in the NCBI database. Thus, the YBS01 strain was preliminarily determined to be Bacillus subtilis (Bacillus subtilis).
SEQ ID NO. 1 is shown below:
gttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaatggctcaccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtg.
2.3 MALDI-TOF-MS protein mass spectrum identification
A small amount of YBS01 single colony is coated on a target plate in a film form; adding 1 mu L of lysate in the mass spectrum sample pretreatment kit, and naturally airing at room temperature; adding 1 mu L of matrix solution in the mass spectrum sample pretreatment kit to cover the sample, and naturally airing at room temperature; and (5) placing the sample target into a mass spectrometer for identification. The identification result shows that the YBS01 strain is bacillus subtilis (Bacillus subtilis), and the protein mass spectrum peak diagram is shown in figure 2.
The applicant utilizes two molecular biological means of 16S rDNA sequencing and MALDI-TOF-MS protein mass spectrum identification system to identify YBS01 strain, and the identification results are identical. And then, combining with colony morphological characteristics of the YBS01 strain, the applicant determines that the strain is bacillus subtilis (Bacillus subtilis) and is named as bacillus subtilis YBS01 (Bacillus subtilis YBS 01).
The applicant has preserved the bacillus subtilis YBS01 (Bacillus subtilis YBS) in China center for type culture collection (CCTCC NO) of university of Wuhan, china on 1 month 13 of 2021: m2021053.
EXAMPLE 3 evaluation of Bacillus subtilis YBS01 enzyme-producing ability
1. Preparation of bacterial liquid
Inoculating the activated bacillus subtilis YBS01 into an LB liquid culture medium, and culturing for 14h at 37 ℃ and 220r/min to obtain a bacterial liquid with the viable bacteria amount of 10 8-109 CFU/ml.
2. Evaluation of protease production ability
The bacillus subtilis YBS01 bacterial liquid is inoculated on an agar medium containing skimmed milk, and is cultured for 48 hours at 37 ℃, so that transparent rings are produced around the bacillus subtilis YBS01, and the diameter of the transparent rings is 25mm. Thus, it is demonstrated that Bacillus subtilis YBS01 can produce certain proteases.
Further, centrifuging the bacillus subtilis YBS01 bacterial liquid at the temperature of 4 ℃ and the speed of 12000rpm for 5min, and taking a supernatant; the protease activity in the fermentation supernatant of Bacillus subtilis YBS01 was measured by the following method.
The results show that the protease activity in the supernatant of the fermentation of the bacillus subtilis YBS01 is 26.6U/ml.
(1) Definition of enzyme Activity
Under certain conditions, the substrate is hydrolyzed per minute to produce an amount of enzyme equivalent to 1 micromolar tyrosine, defined as 1 unit.
(2) Protease activity determination method
① Sucking 1mL of enzyme solution to be detected into a test tube with the thickness of 16 multiplied by 120mm, and preheating for 5min at 37 ℃;
② Adding 1ml casein into enzyme solution without shaking, and reacting at 37deg.C for 10min;
③ Adding 2mL of trichloroacetic acid stop solution, and carrying out vortex oscillation;
④ Standing at room temperature for 5min, and vortex oscillating again;
⑤ Filtering with whatman No.1 filter paper, and measuring 9cm diameter;
⑥ Taking 1ml of enzymatic reaction solution, adding 5mlNa 2CO3 solution, 2 mlfolin-phenol reagent for reacting for 20min at 37 DEG C
⑦ Colorimetric at 680 nm.
⑧ Blank: the ②③ steps are interchanged, the rest are consistent with the sample reaction, and each batch of reaction only needs to be made with 1 blank.
(3) Enzyme activity calculation
Standard curve: ph7.5: mU/mL = 11 x Abs.
Enzyme activity: u=y× (1/1000) ×n.
Wherein Y is the enzyme activity mU;
conversion coefficient from 1/1000-mU to U;
n-dilution factor.
3. Evaluation of Amylase production ability
The bacillus subtilis YBS01 bacterial liquid is inoculated on an agar medium containing starch, and is cultured for 48 hours at 37 ℃, so that transparent rings are produced around the bacillus subtilis YBS01, and the diameter of the transparent rings is 19mm. Thus, it is demonstrated that Bacillus subtilis YBS01 can produce certain amylase.
4. Evaluation of ability to produce starch cellulase
The bacillus subtilis YBS01 bacterial liquid is inoculated on an agar culture medium containing carboxymethyl cellulose, and is cultured for 48 hours at 37 ℃, so that transparent rings are produced around the bacillus subtilis YBS01, and the diameter of the transparent rings is 15mm. Thus, it is demonstrated that bacillus subtilis YBS01 can produce certain cellulases.
EXAMPLE 4 evaluation of deodorizing ability of Bacillus subtilis YBS01
1. Preparation of odor Source
The chicken manure and the peanut shell straw are uniformly mixed according to the mass ratio of 3:1, the water content is regulated to 65 percent, and 200g is taken and placed in a 500ml conical flask for standby.
2. Deodorization experiment
The bacillus subtilis YBS01 bacterial liquid prepared in the example 3 is adjusted to 1 hundred million cfu/ml, inoculated into the odor source according to the volume ratio of 1 percent, and inoculated into LB liquid culture medium with corresponding dosage in a control group. Treatment and control groups were each duplicated in three. The culture was carried out at 37℃for 6 days, and the instantaneous release concentrations (ppm) of ammonia gas and hydrogen sulfide in the odor source were measured at about 12:00 per day by a pump type gas detector, and the average was taken. The specific results are shown in Table 2.
TABLE 2 deodorizing effect of Bacillus subtilis YBS01 on chicken manure
As can be seen from the results in Table 2, compared with the control group, the concentration of ammonia gas and hydrogen sulfide released by chicken manure of the treated group inoculated with Bacillus subtilis YBS01 is greatly reduced every day, and the peak value of odor release is also reduced by 34-47%. Wherein, the release concentration of ammonia is reduced by 70% at most, and the release concentration of hydrogen sulfide is reduced by 78% at most, thus obtaining unexpected technical effects.
3. Detection of maturity and other compost indicators
The decomposition degree is the decomposition degree, and means the degree that organic matters in the compost reach stability after mineralization and humification processes.
According to the new standard of the organic fertilizer NY/T525-2021, the Germination Index (GI) detection index is used for evaluating the decomposition degree of the organic fertilizer, and the index is more than or equal to 70%. The higher the germination index, the lower the toxicity of the decomposed materials to the root system of the crops.
The method for measuring GI is as follows:
Weighing 10g of the sludge sample after decomposing for 6 days, adding 100mL of distilled water, and oscillating for 30min at the temperature of 200rpm and 25 ℃; taking 5mL of filtrate, and adding the filtrate into a 9cm culture dish paved with filter paper; 10 plump cucumber seeds are sown on each dish, and are cultivated for 72 hours at the constant temperature and humidity in a dark place at the temperature of 28 ℃, and distilled water is used as a control, and the cultivation is repeated for 3 times. The germination percentage and the root length of each group of cucumber seeds are counted, and the maturity (germination index) is calculated.
Gi= (germination rate of test group x seed root length)/(germination rate of control group x seed root length) ×100%.
The compost index of the decomposed chicken manure was tested according to the specification of the organic fertilizer NY/T525-2021, and the specific results are shown in Table 3.
TABLE 3 test results of chicken manure compost indicators
| Index (I) | CK | YBS01 treatment group |
| Degree of maturity GI | 44.33％ | 76.31％ |
| Organic matter | 45％ | 40％ |
| Total nutrient (Nitrogen, phosphorus and potassium) | 7.56％ | 8.21％ |
| Moisture content | 85％ | 77％ |
As can be seen from the results in Table 3, the decomposition degree of the chicken manure of the treatment group inoculated with the Bacillus subtilis YBS01 is up to 76.31% compared with that of the control group, the total nutrient content of the chicken manure of the treatment group is improved by 72.1% compared with that of the control group, and the organic matter and the water content are reduced obviously. Therefore, the bacillus subtilis YBS01 provided by the invention can effectively reduce the consumption of nitrogen sources, increase the utilization rate of organic matters, greatly improve the maturity and fertilizer efficiency of chicken manure compost, and has very remarkable effect.
Example 5 application of Bacillus subtilis YBS01 in deodorizing chicken manure compost
1. Preparation of bacillus subtilis YBS01 bacterial powder
Carrying out liquid fermentation on bacillus subtilis YBS01 in a 5-ton fermentation tank, and stopping fermentation when the microscopic examination spore rate reaches more than 90%; centrifuging at 5000rpm for 10min, removing fermentation supernatant, and spray drying the bacterial mud to obtain bacterial powder with viable bacteria amount of 100 hundred million/g.
2. Chicken manure composting material collocation
Uniformly mixing 400 tons of chicken manure and 100 tons of peanut shell straw to prepare four trapezoidal fermentation stacks with the width of 2.5 meters, the height of 1 meter and the length of 60 meters for later use.
3. Deodorization experiment
(1) Experiment site: and 4. A mountain ash-saving tabacco table city, suaeda and biotechnology composting workshop.
(2) Experiment design:
the bacillus subtilis YBS01 bacterial powder is respectively diluted into three doses of 3 hundred million cfu/g,6 hundred million cfu/g and 12 hundred million cfu/g by bran or straw powder. And (3) respectively adding diluted bacterial powder into chicken manure fermentation piles, wherein each treatment corresponds to one pile.
Blank control group: no treatment is carried out;
treatment group 1: 40kg of 3 hundred million cfu/g YBS01 bacteria powder is added;
treatment group 2: 40kg of 6 hundred million cfu/g YBS01 bacteria powder is added;
Treatment group 3: 40kg of 12 hundred million cfu/g YBS01 bacterial powder is added.
6 Points are taken from each pile body, three samples are taken at each point about 12:00 a day, and the temperature of the samples and the instantaneous release concentration (ppm) of ammonia and hydrogen sulfide in the samples are respectively measured, and the average value is obtained. The specific results are shown in Table 4.
TABLE 4 deodorizing effect of Bacillus subtilis YBS01 in chicken manure compost
As can be seen from the data in Table 4, compared with the control group, the release concentration of ammonia and hydrogen sulfide in the chicken manure of each treatment group inoculated with the bacillus subtilis YBS01 bacterial powder is greatly reduced, and the release concentration of odor is obviously reduced along with the increase of the addition amount of the bacterial powder. Wherein the peak value of odor release is reduced by 56% -58%, the release concentration of ammonia gas is reduced by 65% at the highest, the release concentration of hydrogen sulfide is reduced by 60% at the highest, and the chicken manure of the treatment group 2 and the treatment group 3 completely eliminates the emission of hydrogen sulfide on the 3 rd day. Therefore, the bacillus subtilis YBS01 provided by the invention has obvious degradation effect on ammonia and hydrogen sulfide, and unexpected technical effects are achieved.
4. Detection of maturity and other compost indicators
The method for detecting the degree of decomposition was the same as in example 4. The compost index of the decomposed chicken manure is tested according to the specification of the organic fertilizer NY/T525-2021, and the specific result is shown in Table 5.
TABLE 5 test results of chicken manure compost indicators
As can be seen from the results in Table 5, the highest temperature of the chicken manure piles of the treatment group inoculated with the bacillus subtilis YBS01 bacterial powder reaches 64-68 ℃, which is obviously higher than that of the control group (55 ℃), and the average temperature reaches 51-55 ℃ which is also obviously higher than that of the control group (45 ℃). And with the increase of the adding amount of the bacillus subtilis YBS01 bacterial powder, the total nutrient content and the decomposition degree in the processed group chicken manure are obviously improved, and the organic matter and the moisture content are obviously reduced. Wherein, the decomposition degree of the chicken manure in the treatment group 3 is improved by 170.7 percent compared with that in the control group, which is as high as 76.21 percent, thus obtaining unexpected technical effect.
The result shows that the bacillus subtilis YBS01 provided by the invention can obviously improve the chicken manure composting temperature, is beneficial to quickly killing pathogenic microorganisms in compost and quickens the decomposition process; meanwhile, the strain can effectively reduce the consumption of nitrogen sources, increase the utilization rate of organic matters, greatly improve the decomposition degree and fertilizer efficiency of compost and has very obvious effect.
Example 6 application of Bacillus subtilis YBS01 in deodorization of cow dung compost
1. Cow dung compost material collocation
100 Tons of fresh cow dung and 20 tons of mushroom residues are uniformly mixed to prepare four trapezoidal fermentation piles with the width of 2.5 meters, the height of 1 meter and the length of 30 meters for standby.
2. Compost deodorization experiment
(1) Experiment site: hongtai valley fermentation workshop in Jinzhong of Shanxi province.
(2) Experiment design:
the bacillus subtilis YBS01 bacterial powder is respectively diluted into three doses of 3 hundred million cfu/g,6 hundred million cfu/g and 12 hundred million cfu/g by bran or straw powder. And (3) respectively adding diluted bacterial powder into cow dung fermentation stacks, wherein each treatment corresponds to one stack.
Blank control group: no treatment is carried out;
treatment group 1: 30kg of 3 hundred million cfu/g YBS01 bacteria powder is added;
Treatment group 2: 30kg of 6 hundred million cfu/g YBS01 bacteria powder is added;
Treatment group 3: 30kg of 12 hundred million cfu/g YBS01 bacteria powder is added.
Turning the stack every 2 days, taking 6 points from each stack body, taking three samples at each point about 12:00 a day, respectively measuring the temperature of the samples and the instantaneous release concentration (ppm) of ammonia and hydrogen sulfide in the samples, and taking an average value. The specific results are shown in Table 6.
TABLE 6 deodorizing effect of Bacillus subtilis YBS01 in cow dung compost
As can be seen from the data in Table 6, compared with the control group, the release concentration of cow dung hydrogen sulfide and ammonia gas in each treatment group inoculated with the bacillus subtilis YBS01 bacterial powder is greatly reduced, and the release concentration of odor is obviously reduced along with the increase of dosage. Wherein, the peak value of ammonia gas release is reduced by 79% -88%, and the release concentration of ammonia gas is reduced by 88% at most; the instantaneous release amount of the hydrogen sulfide in the cow dung in the treatment group 1 is reduced to 0 on the 4 th day of fermentation and is 2 days earlier than that in the control group, while the instantaneous release amount of the hydrogen sulfide in the treatment group 2 and the treatment group 3 is reduced to 0 on the 3 rd day of fermentation and is 3 days earlier than that in the control group. Therefore, the bacillus subtilis YBS01 provided by the invention has obvious degradation effect on ammonia and hydrogen sulfide, and unexpected technical effects are achieved.
3. Detection of maturity and other compost indicators
The composting degree test method was the same as in example 3, and the composting index of the above-mentioned decomposed materials was tested according to the rules of organic fertilizer NY/T525-2021, and the specific results are shown in Table 7.
TABLE 7 compost indicator assay results
As can be seen from the results in Table 7, the highest temperature of the cow dung piles of the treatment group inoculated with the bacillus subtilis YBS01 bacterial powder reaches 66-69 ℃ in the composting process, which is obviously higher than that of the control group (65 ℃), and the average temperature reaches 54-56 ℃ and is also obviously higher than that of the control group (42 ℃). And with the increase of the adding amount of the bacillus subtilis YBS01 bacterial powder, the total nutrient content and the decomposition degree in the sludge compost of the treatment group are obviously improved, and the organic matter and the moisture content are obviously reduced. The decomposition degree of the cow dung compost in the treatment group 3 is improved by 64 percent and is as high as 80 percent compared with that in the control group, and unexpected technical effects are achieved.
The result shows that the bacillus subtilis YBS01 provided by the invention can raise the temperature of cow dung compost, is beneficial to quickly killing pathogenic microorganisms in the compost and quickens the decomposition process; meanwhile, the strain can effectively reduce the consumption of nitrogen sources, increase the utilization rate of organic matters, greatly improve the decomposition degree and fertilizer efficiency of compost and has very obvious effect.
Example 7 application of Bacillus subtilis YBS01 in deodorization of pig manure compost
1. Pig manure composting material collocation
Uniformly mixing 40 tons of pig manure and 10 tons of peanut shell straw to prepare four trapezoidal fermentation stacks with the width of 2.5 meters, the height of 1 meter and the length of 10 meters for later use.
2. Compost deodorization experiment
(1) Experiment site: a Sanhe biological fermentation workshop in Laiyang city of the tobacco stage of Shandong province.
(2) Experiment design:
The bacillus subtilis YBS01 bacterial powder is respectively diluted into three doses of 3 hundred million cfu/g,6 hundred million cfu/g and 12 hundred million cfu/g by bran or straw powder. And (3) respectively adding diluted bacterial powder into pig manure fermentation piles, wherein each treatment corresponds to one pile.
Blank control group: no treatment is carried out;
Treatment group 1: 12.5kg of 3 hundred million cfu/g YBS01 bacteria powder is added;
treatment group 2: 12.5kg of 6 hundred million cfu/g YBS01 bacteria powder is added;
Treatment group 3: 12.5kg of 12 hundred million cfu/g YBS01 bacteria powder is added.
Turning once a day, taking 6 points from each pile body, taking three samples at each point about 12:00 a day, respectively measuring the temperature of the samples and the instantaneous release concentration (ppm) of ammonia and hydrogen sulfide in the samples, and taking an average value. The specific results are shown in Table 8.
Table 8 deodorizing effect of Bacillus subtilis YBS01 in pig manure compost
As can be seen from the data in Table 8, compared with the control group, the release concentration of the pig manure hydrogen sulfide and ammonia gas in each treatment group inoculated with the bacillus subtilis YBS01 strain powder is greatly reduced, and the release concentration of the odor is obviously reduced along with the increase of the dosage. Wherein, the peak value of ammonia gas release is reduced by 55% -71%, and the maximum concentration of ammonia gas release is reduced by 71%; the peak value of the release of the hydrogen sulfide is reduced by 76% -80%, and the release concentration of the hydrogen sulfide is reduced by 97% at most; treatment group 1 and treatment group 2 pig manure had completely eliminated hydrogen sulfide emissions on day 6 of fermentation, and treatment group 3 had completely eliminated hydrogen sulfide emissions on day 5 of fermentation, significantly earlier than the control group. Therefore, the bacillus subtilis YBS01 provided by the invention has obvious degradation effect on ammonia and hydrogen sulfide in pig manure, and unexpected technical effects are achieved.
4. Detection of maturity and other compost indicators
The composting degree test method was the same as in example 3, and the composting index of the above-mentioned decomposed materials was tested according to the rules of organic fertilizer NY/T525-2021, and the specific results are shown in Table 9.
TABLE 9 compost indicator assay results
As can be seen from the results in Table 9, the highest temperature of the pig manure bulk of the treatment group inoculated with the bacillus subtilis YBS01 strain powder reaches 66-69 ℃ in the composting process, which is significantly higher than that of the control group (60 ℃) and the average temperature reaches 56-57 ℃ which is also significantly higher than that of the control group (46 ℃). And with the increase of the adding amount of the bacillus subtilis YBS01 bacterial powder, the total nutrient content and the decomposition degree in the treated pig manure compost are obviously improved, and the organic matter and the moisture content are obviously reduced. The decomposition degree of the pig manure compost in the treatment group 3 is improved by 130 percent and is as high as 78 percent compared with that in the control group, and unexpected technical effects are achieved.
The result shows that the bacillus subtilis YBS01 provided by the invention can raise the temperature of pig manure compost, is beneficial to quickly killing pathogenic microorganisms in compost and quickens the decomposition process; meanwhile, the strain can effectively reduce the consumption of nitrogen sources, increase the utilization rate of organic matters, greatly improve the decomposition degree and fertilizer efficiency of pig manure compost, and has very obvious effect.
EXAMPLE 8 evaluation of deodorization Capacity of Bacillus subtilis YBS01 formulated with decomposed inoculant
1. Preparation of odor Source
The chicken manure and the peanut shell straw are uniformly mixed according to the mass ratio of 4:1, the water content is regulated to 65%, and 200g of each treatment is placed in a 500ml conical flask for standby.
2. Deodorization experiment
The bacillus subtilis YBS01 bacterial liquid prepared in the example 3 is adjusted to 1 hundred million cfu/ml, and inoculated into the odor source according to different dosages.
(1) Blank control group: inoculating LB liquid culture medium according to the volume ratio of 1%;
(2) Inoculant control group: inoculating 0.1g of a rotten inoculant (50 hundred million cfu/g);
(3) YBS01 control group: inoculating bacillus subtilis YBS01 bacterial liquid (1 hundred million cfu/ml) according to the volume ratio of 1%;
(4) YBS01 complex: bacillus subtilis YBS01 bacterial liquid (1 hundred million cfu/ml) is inoculated according to the volume ratio of 1 percent, and 0.1g of decomposed inoculant (50 hundred million cfu/g) is inoculated at the same time.
Three replicates were made for each of the treatment and control groups. The samples were incubated at 37℃for 7 days, and the instantaneous release concentrations (ppm) of ammonia and hydrogen sulfide from the odor source were measured at about 12:00 a day using a pump gas detector, and averaged. The specific results are shown in Table 10.
TABLE 10 deodorizing effect of different treatments on chicken manure
As can be seen from the results in table 10, the release amount of ammonia gas in the inoculant control group is reduced, but the release amount of hydrogen sulfide is obviously improved, which indicates that the addition of the decomposition agent can further improve the release amount of hydrogen sulfide in chicken manure; the bacillus subtilis YBS01 can obviously reduce the release of ammonia and hydrogen sulfide no matter being single bacteria or being compounded with a decomposed inoculant, obviously reduce the peak value of the release of odor, and enable the release of the odor source to reach a lower level in a shorter time. Wherein, the release peak value of the ammonia gas and the hydrogen sulfide of the chicken manure of the control group added with the bacillus subtilis YBS01 is reduced by 44-62%, the release concentration of the ammonia gas is reduced by 66% at most, and the release concentration of the hydrogen sulfide is reduced by 89% at most; meanwhile, the release peak value of ammonia gas and hydrogen sulfide of the compound chicken manure added with the bacillus subtilis YBS01 and the decomposed inoculant is reduced by 45-55%, the release concentration of ammonia gas is reduced by 48% at most, and the release concentration of hydrogen sulfide is reduced by 80% at most, so that the effect is very remarkable. 4. Detection of maturity and other compost indicators
The method for detecting the degree of decomposition was the same as in example 4. The compost index of the decomposed chicken manure was tested according to the specification of the organic fertilizer NY/T525-2021, and the specific results are shown in Table 11.
Table 11 chicken manure compost indicator test results
| Treatment of | Blank control group | Inoculant control group | YBS01 control group | YBS01 compound group |
| Degree of maturity (GI) | 53.62％ | 76.31％ | 64.94％ | 90.08％ |
| Organic matter | 42％ | 38％ | 39％ | 35％ |
| Total nutrient (Nitrogen, phosphorus and potassium) | 8.18％ | 8.03％ | 8.20％ | 8.21％ |
| Moisture content | 84％ | 72％ | 75％ | 70％ |
As can be seen from the results of Table 11, in the fermentation period of 6 days, the bacillus subtilis YBS01 provided by the invention can obviously improve the maturity of chicken manure by up to 90% no matter being single bacteria or being compounded with the maturity inoculant, and has the advantages of quickened organic matter decomposition, reduced nutrient consumption, large water evaporation amount, contribution to quickening the composting process and shortening the composting period.
Example 9 application of Bacillus subtilis YBS01 and compost rotten inoculant in chicken manure composting
1. Experiment site: and 4. A mountain ash-saving tabacco table city, suaeda and biotechnology composting workshop.
2. Sludge composting materials are matched:
Uniformly mixing 400 tons of chicken manure and 100 tons of peanut shell straw to prepare four trapezoidal fermentation stacks with the width of 2.5 meters, the height of 1 meter and the length of 60 meters for later use.
3. Deodorization experiment:
the bacillus subtilis YBS01 bacterial powder is respectively diluted into three doses of 3 hundred million cfu/g,6 hundred million cfu/g and 12 hundred million cfu/g by bran or straw powder. And (3) respectively adding diluted bacterial powder into chicken manure fermentation piles, wherein each treatment corresponds to one pile.
Blank control group: 40kg of decomposed inoculant is added, and YBS01 bacterial powder is not added;
Treatment group 1: 40kg of decomposed inoculant and 40kg of 3 hundred million cfu/g YBS01 bacteria powder are added;
treatment group 2: 40kg of decomposed inoculant and 40kg of 6 hundred million cfu/g YBS01 bacteria powder are added;
Treatment group 3: 40kg of decomposed inoculant and 40kg of 12 hundred million cfu/g YBS01 bacteria powder are added.
6 Points are taken from each pile body, three samples are taken at each point about 12:00 a day, and the temperature of the samples and the instantaneous release concentration (ppm) of ammonia and hydrogen sulfide in the samples are respectively measured, and the average value is obtained. The specific results are shown in Table 12.
Table 12 deodorizing effect of Bacillus subtilis YBS01 compound inoculant in chicken manure compost
As can be seen from the data in Table 12, compared with the control group, the release concentrations of the chicken manure hydrogen sulfide and ammonia gas in the treatment group added with the decomposed inoculant and the bacillus subtilis YBS01 bacterial powder are both greatly reduced, and the peak value of odor release is also remarkably reduced. Wherein, the release concentration of ammonia is reduced by 60% at most, the release concentration of hydrogen sulfide is reduced by 83% at most, and the release of hydrogen sulfide is completely eliminated by the chicken manure of the treatment group 1 on the 5 th day, the amounts of hydrogen sulfide of the treatment groups 2 and 3 on the 3 rd day are undetectable, and the control group is still detectable on the 6 th day. Therefore, the bacillus subtilis YBS01 provided by the invention has obvious degradation effect on hydrogen sulfide and ammonia gas, can increase the deodorizing function of the decomposing agent, and achieves unexpected technical effects.
4. Decomposition degree detection and other compost index detection
The composting degree test method was the same as in example 3, and the composting index of the above-mentioned decomposed materials was tested according to the rules of organic fertilizer NY/T525-2021, and the specific results are shown in Table 13.
TABLE 13 compost indicator assay results
| Treatment of | CK | Treatment group 1 | Treatment group 2 | Treatment group 3 |
| Highest temperature (DEG C) | 70℃ | 72℃ | 74℃ | 78℃ |
| Average temperature DEG C | 61℃ | 62℃ | 62℃ | 65℃ |
| Degree of maturity (GI) | 79.65％ | 88.38％ | 105.66％ | 121.23％ |
| Organic matter | 45％ | 45％ | 43％ | 40％ |
| Total nutrient (Nitrogen, phosphorus and potassium) | 7.98％ | 7.92％ | 8.14％ | 8.17％ |
| Moisture content | 46％ | 44％ | 44％ | 42％ |
As can be seen from the results in Table 13, the combination of the Bacillus subtilis YBS01 and the decomposed inoculant can obviously improve the effect of the decomposed inoculant compared with the control group, the decomposition degree of the chicken manure of the treatment group is obviously improved, and the decomposition degree is further increased by 121.23 percent and is improved by 52.2 percent compared with the control group along with the increase of the addition amount of the Bacillus subtilis YBS 01. In addition, the bacillus subtilis YBS01 can also obviously improve the composting temperature of chicken manure, and compared with a control group, the highest composting temperature and the average composting temperature of the treatment group are respectively improved by 8 ℃ and 4 ℃, the decomposing period of 3 days is shortened, and unexpected technical effects are achieved.
In conclusion, the bacillus subtilis YBS01 provided by the invention can be singly used, can also be compounded with any one or two or more of bacillus, aspergillus, saccharomycetes and lactobacillus or a decomposed inoculant, is widely applied to fecal composting, can obviously reduce the emission of odor gases such as ammonia gas, hydrogen sulfide and the like, can obviously improve the composting temperature, accelerates the decomposition process, shortens the composting period, improves the decomposition degree and the fertilizer efficiency, and has wide application prospect.
Sequence listing
<110> Shandong Uygur biotechnology Co., ltd
Shandong Kangdi En Biotech Co., ltd
<120> Bacillus subtilis and application thereof in deodorization of fecal compost
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1355
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 1
gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg ggataactcc 60
gggaaaccgg ggctaatacc ggatggttgt ttgaaccgca tggttcaaac ataaaaggtg 120
gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaatggc 180
tcaccaaggc aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga 240
cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 300
gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgttaggg 360
aagaacaagt accgttcgaa tagggcggta ccttgacggt acctaaccag aaagccacgg 420
ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggaattattg 480
ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg 540
ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt 600
agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg 660
taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 720
acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa 780
cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 840
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 900
aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac 960
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020
gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg 1080
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1140
acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac 1200
aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta 1260
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320
cacaccacga gagtttgtaa cacccgaagt cggtg 1355
Claims (9)
1. The bacillus subtilis (Bacillus subtilis) is characterized in that the collection number of the bacillus subtilis is CCTCC NO: m2021053.
2. Use of bacillus subtilis according to claim 1 for biological deodorization.
3. The use of bacillus subtilis according to claim 1 in compost production.
4. A biological deodorant, characterized in that it comprises the bacillus subtilis according to claim 1.
5. The biological deodorant according to claim 4, further comprising any one or a combination of two or more of bacillus, aspergillus, yeast, and lactic acid bacteria.
6. The biological deodorant according to claim 5, wherein the living bacterial amount of bacillus subtilis in the biological deodorant is at least 10 8 CFU/g.
7. Use of a biological deodorant according to any one of claims 4-6 for the deodorization of manure compost.
8. The use according to claim 7, wherein the faeces are animal faeces.
9. The use according to claim 8, wherein the manure is any one of chicken manure, duck manure, pig manure, cow manure, sheep manure.
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| CN107325992A (en) * | 2017-08-31 | 2017-11-07 | 成都友益佳环保设备工程有限公司 | A kind of bacillus subtilis strain and its application |
| CN110184218A (en) * | 2019-05-29 | 2019-08-30 | 华中农业大学 | A kind of composite bacteria agent and application based on bacillus subtilis BS1 |
| CN113151034A (en) * | 2020-12-31 | 2021-07-23 | 上海康地恩生物科技有限公司 | Bacillus subtilis strain for deodorizing livestock and poultry breeding environment |
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| CN107325992A (en) * | 2017-08-31 | 2017-11-07 | 成都友益佳环保设备工程有限公司 | A kind of bacillus subtilis strain and its application |
| CN110184218A (en) * | 2019-05-29 | 2019-08-30 | 华中农业大学 | A kind of composite bacteria agent and application based on bacillus subtilis BS1 |
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