CN116676212B - Brevibacillus aydinogluensis PMBT001 strain and application thereof - Google Patents
Brevibacillus aydinogluensis PMBT001 strain and application thereof Download PDFInfo
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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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/13—Brevibacterium
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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 a strain Brevibacillus aydinogluensis PMBT001 and application thereof, wherein the preservation number is GDMCC.No:63294; the collection unit is the department of microbiology of the academy of sciences of Guangdong (collection of microbiological bacterial strains of Guangdong), the collection unit address: the Guangdong province, guangzhou City, first, china, no. 100, no. 59, 5, and Guangdong province microbiological institute, with a storage date of 2022, 03 and 27. The gene sequence of Brevibacillus aydinogluensis PMBT001 is shown in SEQ ID NO. 1. The invention takes panda feces as a separation source, adopts high-temperature separation to screen out a strain of thermophilic bacteria with three enzyme activities for aerobic composting fermentation, has the proper growth temperature range of 40-70 ℃, has higher cellulase activity and protease activity, and can reach the relative enzyme activity of cellulose of 21.7 mu mol/mL, the relative enzyme activity of protease of 0.26 mu mol/mL and the relative enzyme activity of lipase of 10.00 mu mol/mL at the high temperature of 50 ℃.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Brevibacillus aydinogluensis PMBT001 strain and application thereof.
Background
At present, high-temperature aerobic composting treatment is an important way for solving the problem of solid waste, and has the advantages of environment protection, raw material and energy conservation, low investment, low running cost and the like. The existing research shows that a long-time high-temperature period exists in the composting process, most parasites, ova, spores, pathogenic bacteria and the like in the compost are killed in the high-temperature period of the compost, and the high-temperature period is not only the main period of decomposition of organic macromolecules, but also the important period of ensuring innocuity of the compost. Microorganisms are extremely susceptible to high temperature factors during this period, population numbers and activities thereof are generally reduced, and rapid decomposition of organic substances is inhibited. A great deal of researches show that the temperature of the pile body is controlled between 45 ℃ and 65 ℃, and especially the optimal temperature for degrading macromolecular substances such as lignocellulose and the like at 55 ℃ to 60 ℃. The research on the microorganism and the activity characteristic thereof in the temperature range has important significance for improving the composting technology. Therefore, the study of the thermophilic bacteria has become a hot spot. The high temperature bacteria are inoculated in the composting treatment, so that the quantity of high Wen Qihui organisms can be increased, the rapid conversion of certain refractory organic matters can be promoted, and the composting efficiency can be further effectively improved. Research shows that adding high temperature bacteria can raise the conversion of organic matter, speed up the composting process, reduce the exhaust of odorous gas, stabilize the compost product and raise the compost quality.
Agricultural solid waste, mainly crop straw, livestock manure and dead livestock carcasses, contains a large amount of cellulose, protein, fat substances and the like, has complex components, and is difficult to fully utilize or be directly used as carbon source substances by most microorganisms for conversion and utilization. Researches show that the cellulose degrading bacteria can effectively improve the composting temperature, accelerate the composting process, accelerate the degradation of straw and improve the content of humus in the compost when applied to the compost, thereby improving the quality of the compost; the protein and fat degrading bacteria are applied to compost, can effectively degrade dead livestock and poultry carcasses and livestock and poultry manure, eliminate compost odor and activate nutrients, thereby effectively killing pathogenic microorganisms and achieving the harmless requirement. Therefore, high-temperature resistant efficient cellulose, protein and fat degrading bacteria are screened and inoculated into the compost, so that the compost can be promoted to be decomposed, and the quality of a compost product can be improved. The research on aerobic composting of high temperature strains is very active nowadays, but the previous research on high temperature strains is mainly focused on amylase activity, antibiotic production and the like, and the research on high temperature strains with cellulase, protease and lipase degradation capability is very little.
The bacterial culture period is short, the growth speed is high, the low-cost nitrogen source and carbon source can be used as energy sources for producing the enzyme, the enzyme expression quantity is relatively high, the enzyme is relatively easy to obtain, the thermal stability of the enzyme is good, and the prokaryotic expression control system is relatively simple and convenient. Thus, bacteria have their unique advantage in degrading cellulose, proteins and fats.
Disclosure of Invention
The invention aims to solve the technical problem of providing Brevibacillus aydinogluensis PMBT001 strain and application thereof, wherein Brevibacillus aydinogluensis PMBT001 obtained by screening is a high-temperature bacterium capable of producing cellulase, protease and lipase, and the strain is applied to aerobic composting, so that the fecal composting process can be accelerated, and the nutrient content can be increased.
In order to solve the problems in the prior art, the invention adopts the following technical scheme:
in a first aspect, the invention provides a strain Brevibacillus aydinogluensis PMBT001 having a deposit number gdmcc.no:63294; the collection unit is the department of microbiology of the academy of sciences of Guangdong (collection of microbiological bacterial strains of Guangdong), the collection unit address: the Guangdong province, guangzhou City, first, china, no. 100, no. 59, 5, and Guangdong province microbiological institute, with a storage date of 2022, 03 and 27.
Further, the gene sequence of Brevibacillus aydinogluensis PMBT001 is shown in SEQ ID NO. 1.
In a second aspect, the invention provides the use of Brevibacillus aydinogluensis PMBT0011 in the preparation of a microbial agent.
In a third aspect, the present invention provides a microbial agent comprising Brevibacillus aydinogluensis PMBT001 as described in the first aspect.
Further, the number of viable bacteria of the Brevibacillus aydinogluensis PMBT001 microbial agent is 1.0X10 8 -5.0×10 8 CFU/g。
Preferably, the number of viable bacteria of the Brevibacillus aydinogluensis PMBT001 microbial agent is 1.3X10 8 CFU/g, pH value is 6.0-8.0.
In a fourth aspect, the present invention provides a method for preparing the microbial agent according to the third aspect, comprising the steps of:
inoculating Brevibacillus aydinogluensis PMBT001 into TSB culture medium, and culturing at 50-60deg.C and 160-200r/min for 1 day to obtain seed solution; inoculating the seed solution into a fermentation culture medium according to an inoculum size of 5% (v/v), and performing shake culture at 50-60 ℃ for 1-2 days at 160-200r/min to obtain a microbial agent;
the components of the fermentation medium are as follows: 7g of protein pulse, 1g of beef extract, 5g of sodium chloride, 10g of glucose and 1000mL of distilled water.
In a fifth aspect, the present invention provides the use of Brevibacillus aydinogluensis PMBT001 as set forth in the first aspect or the microbial agent as set forth in the third aspect in the preparation of a microbial fertilizer.
In a sixth aspect, the present invention provides a microbial fertilizer comprising Brevibacillus aydinogluensis PMBT001 as described in the first aspect or the microbial agent as described in the third aspect.
In a seventh aspect, the present invention provides the use of Brevibacillus aydinogluensis PMBT001 as defined in the first aspect, the microbial agent as defined in the third aspect, or the microbial fertilizer as defined in the sixth aspect in the high-temperature aerobic composting of manure.
In an eighth aspect, the present invention provides a high temperature aerobic composting process comprising the steps of:
s1: uniformly mixing fresh manure with urea, wood chips and wood shavings to form mixed manure, wherein the mixed manure finally contains 0.05-0.4% of urea, 20-30% of wood chips and wood shavings by mass percent;
s2: the microbial agent described in the third aspect is added according to 0.3-1.0% of the volume of the mixed excrement, and then fermentation is carried out for 10-30 days by adopting a stacking method.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes panda feces as a separation source, adopts high-temperature separation to screen out a bacterial strain Brevibacillus aydinogluensis PMBT001 with three enzyme activities for aerobic composting fermentation, has the temperature range of 40-70 ℃ suitable for growth, and has the functions of producing cellulase, protease and lipase. Has higher cellulase activity and protease activity, can reach the relative enzyme activity of cellulose of 21.7 mu mol/mL and the relative enzyme activity of protease of 0.26 mu mol/mL and the relative enzyme activity of lipase of 10.00 mu mol/mL at the high temperature of 50 ℃. The bacterial strain PMBT001 is applied to cattle manure composting in Zhaojuan county to obtain the effect of promoting the improvement of the composting efficiency and the utilization of organic wastes, and the screening of the high-temperature enzyme-producing strain provides dominant strains for the research and development of manure degradation microbial agents, is beneficial to solving the problems of air pollution, soil pollution, water pollution and the like caused in the composting process, activates nutrients, improves the composting effect and has better application potential for the treatment of the organic wastes.
The preservation information of Brevibacillus aydinogluensis PMBT001 of the invention is as follows:
preservation number: gdmcc.no:63294;
taxonomic name: brevibacillus aydinogluensis;
preservation unit: the institute of microbiology (collection of microbiology, cantonese province);
deposit unit address: building 5 of Guangzhou City of Guangdong, first, china, no. 100, institute of Highway, 59, and microbiological institute of Guangdong;
preservation date: 2022, 03, 27.
Drawings
FIG. 1 is a microscopic image of the morphology and cells of Brevibacillus aydinogluensis PMBT001 colonies on TSA medium;
FIG. 2 is a Brevibacillus aydinogluensis PMBT001 phylogenetic tree constructed based on the 16S rRNA gene sequence;
FIG. 3 is a Brevibacillus aydinogluensis PMBT001 enzyme function assay in which (left) cellulose degradation primary screening results; (ii) preliminary screening results of protein degradation; (right) results of preliminary screening for fat degradation;
FIG. 4 is a graph of compost sample temperature variation;
FIG. 5 is a compost sample C/N variation;
FIG. 6 is a graph showing the variation of seed Germination (GI) of compost samples;
FIG. 7 is a graph of compost sample pH change;
FIG. 8 is a sample of total phosphorus from a compost;
FIG. 9 is a total potassium variation of a compost sample;
FIG. 10 shows the dynamic change of bacterial communities during composting, wherein A is PLS-DA analysis, B is a column of portal-level species abundance, and C is a column of genus-level species abundance.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In this application:
trypticase soy agar medium (TSA medium): 15g of tryptone, 5g of soybean peptone, 5g of NaCl, 20g of agar and 1000mL of distilled water.
Trypticase soytone liquid medium (TSB medium): tryptone 17g, soytone 3g, naCl 5g, K 2 HPO 4 2.5g, glucose 2.5g, agar 20g, distilled water 1000mL.
Congo red solid medium: CMC-Na 2.0g, KH 2 PO 4 0.5g，MgSO 4 ·7H 2 O 0.25g，(NH 4 ) 2 SO 4 1.0g, congo red 0.2g, agar 20g, distilled water 1000mL.
The carbon source is a rescreening culture medium of CMC-Na: CMC-Na 5.0g, KH 2 PO 4 1.0g，NaNO 3 3.0g，KCl 0.5g，MgSO 4 ·7H 2 O 0.5g，FeCl 3 ·6H 2 O0.01 g, distilled water 1000mL.
Skim milk solid medium: 2.5g of yeast extract, 2g of peptone, 1.5g of acid hydrolyzed casein, 1.5g of casein, 40g of skim milk, 20g of agar, 1g of glucose and 1000mL of distilled water.
Protease seed medium: 10g of glucose, 20g of yeast powder, 5g of NaCl and K 2 HPO 4 1g，MgSO 4 0.4g, pH7.0, and 1000mL of distilled water.
Protease basal fermentation medium: glucose 5g, yeast powder 10g, (NH) 4 ) 2 SO 4 1g，CaCl 2 1g，NaCl 1g，KH 2 PO 4 0.5g，MgSO 4 0.3g, pH7.0, 1000mL of distilled water.
Victoria blue B solid medium: protein pulse 10g, beef extract 3g, sodium chloride 5g, agar 20g, olive oil 25mL, distilled water 1000mL, victoria blue (4 mg/100 mL).
Example 1
The embodiment provides a microbial agent, and the preparation method of the microbial agent is as follows:
inoculating Brevibacillus aydinogluensis PMBT001 plate streak to TSA culture medium, picking single colony, inoculating to TSB culture medium, and culturing at 50-60deg.C and 160-200r/min for 1 day to obtain seed solution; the seed solution is mixed according to 5%(v/v) inoculating the inoculum size into fermentation medium, and shake culturing at 50-60deg.C and 160-200r/min for 1-2 days to obtain microbial agent with effective viable count of 1.3X10 8 CFU/g, pH value is 6.0-8.0.
The components of the fermentation medium are as follows: 5-10g of protein pulse, 1g of beef extract, 5g of sodium chloride, 10g of glucose and 1000mL of distilled water.
Example 2
The embodiment provides a high-temperature aerobic composting treatment method, which comprises the following steps:
s1: fresh cow dung from a large-scale national farm in Zhaoju county, sichuan province, china is uniformly mixed with auxiliary materials of urea, wood dust and wood shavings to form mixed excrement, and the mixed excrement finally contains urea with the mass fraction of 0.2% and wood dust and wood shavings with the mass fraction of 25%. The mixed excrement contains urea with the mass fraction of 0.2 percent and wood chips and shavings with the mass fraction of 25 percent, which is helpful for adjusting the carbon-nitrogen ratio.
S2: the microbial agent described in example 1 was added at 0.5% of the mixed stool volume.
S3: the mixed manure is fermented for 14 days by adopting a stacking method in a manure treatment plant. Thoroughly mixing the compost twice in the whole composting process, and turning the compost for the first time when the stacking temperature reaches more than 50 ℃; when the temperature is up to 60 ℃, the second turning is carried out to maintain the aerobic condition and uniformity and promote the degradation of substances.
Experimental example 1 isolation and purification of high temperature bacterium PMBT001
1.1 separation of aerobic composting high temperature bacteria
Panda manure compost (compost temperature is more than or equal to 50 ℃) is collected at panda centers at a river weir of Sichuan province in 2021, is filled into a sterile sampling bag, sealed, is stored in an ice box, and is transported back to a laboratory for bacterial separation treatment within 24 hours.
Weighing fresh compost sample, shaking in sterile water for 1h, and preparing into 10 by 10-fold dilution method -3 -10 -6 Taking 100 mu L of each diluted gradient bacterial suspension, respectively, carrying out separation culture of high-temperature bacteria by using a trypticase soy agar medium (TSA) by a flat plate coating method, wherein the culture temperature is 50+/-2 ℃ and the culture time is 48 hours, and observing the growth condition of colonies during the culture periodThe condition is recorded; under this culture condition, colonies grow rapidly, and individual colonies with larger colonies are transferred into Trypticase Soy Agar (TSA) and purified by dilution plate streaking until purified strains are obtained. 1-2 single colonies were picked, the purified strain was transferred to trypticase soytone liquid medium (TSB), and 50% glycerol was added to store in a-80℃refrigerator.
1.2 identification of strains
1.2.1 Strain morphology
1-2 single colonies were picked, inoculated on TSA medium, placed at 50℃for 1 day of culture, and the colony morphology of the strain was observed.
1.2.2 molecular characterization of strains
(1) Reagent: sterile double distilled water, mix (Tiangen Biochemical technology Beijing Co., ltd.).
(2) Primer(s)
8-27F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2) and
1492-1523R：5’-TAC-GACTTAACCCCAATCGC-3’(SEQ ID NO.3)。
(3) Bacterial DNA extraction
Taking a few thalli in an aseptic 1.5mL Eppendorf tube, adding 500 μl of aseptic double distilled water, freezing a centrifuge tube by adopting liquid nitrogen, placing in a water bath at 99 ℃ for 5min, taking out, swirling for 30s, repeating the above operation for 1 time, centrifuging at 12000r/min, taking the supernatant as a template, and preserving at-20 ℃;1% agarose gel electrophoresis detection.
(4) Bacterial 16S rRNA gene amplification
16S rRNA gene amplification conditions: general primers 8-27F: and 1492-1523R to carry out gene amplification on the extracted bacterial RNA, carrying out pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1.5min,30 cycles and total extension at 72 ℃ for 20min. The PCR product was purified by Shanghai Ind EZ Spin Column PCR Product Purification Kit UNlQ-1 column PCR product purification kit (SK 1142-N) according to the manual, and the purified product was sent to the Bioengineering Co.Ltd for sequencing.
(5) 16S rRNA gene sequence analysis and phylogenetic tree construction
And (3) carrying out similarity search on the sequenced sequences in NCBI by using BLAST software, selecting the 16S rRNA gene sequence of the strain with highest similarity as a reference sequence, carrying out multi-sequence comparison and analysis by using Clustal X software, constructing a phylogenetic tree by using an N-J method through MEGA 7.0 software, and determining the classification status of bacteria.
1.3 experimental results
Bacterial PMBT001 is separated from panda fecal compost, and after culturing for 1d at 50 ℃ on a TSA culture medium, the bacterial colony is flat and round, smooth in surface, thin in edge, opaque, basically neat, milky in color and easy to pick. Gram staining is negative, aerobic, and the thallus is rod-shaped, has spores, and has one end enlarged, and the thallus size is 2-4nm, as shown in figure 1.
The strain PMBT001 was subjected to 16S rRNA sequence determination, BLAST homology alignment was performed in NCBI database, and the similarity of the strain with Brevibacillus aydinogluensis reached 100%. And combining the morphological characteristics, physiological and biochemical characteristics, 16S rRNA sequence homology analysis and other experimental results, and identifying PMBT001 as Brevibacillus aydinogluensis, as shown in FIG. 2.
The sequence of the 16S rRNA gene with the sequence length of Brevibacillus aydinogluensis PMBT [27F ] being 1251bp,Brevibacillus aydinogluensis PMBT001 is shown in SEQ ID NO. 1.
Experimental example 2 determination of growth temperature and enzyme production function of high temperature bacterium PMBT001
2.1 temperature growth Range determination
And (3) inoculating 10 mu L of the glycerol-preserved strain on a TSA culture medium for activation culture, respectively placing the strain at the temperature of 30-80 ℃ for culturing for 48 hours, observing and recording the growth condition, and taking 5 ℃ as a unit according to the measurement result, and further accurately measuring the highest growth temperature and the lowest growth temperature of the strain.
2.2 measurement of cellulose degradation Capacity
2.2.1 cellulose degradation Capacity Primary screening
3-5 single bacterial colonies of activated bacteria are selected, 3mL of sterile water is added to prepare bacterial suspension, 50 mu L of bacterial liquid is evenly coated on a TSA culture medium, the TSA culture medium is placed in a biochemical incubator at 50 ℃ for culture, strain growth on the surface of the culture medium is observed to be even, the strain which grows evenly and the culture medium are punched together with the strain to prepare bacterial cakes by a puncher with the diameter of 5mm and inoculated on a Congo red solid culture medium, the culture medium is placed in the biochemical incubator at 50 ℃ for 3-5d, whether transparent hydrolysis rings are generated at the bacteria-inoculated position of the culture medium is observed, the diameter of the hydrolysis rings is measured, and 3 repetitions are set.
If the strain cannot produce hydrolysis circles on Congo red culture medium and the diameter of the hydrolysis circles is not more than 5mm, the strain is regarded as not having cellulose degradation capability and is marked as-; if the diameter of the hydrolytic circle generated by the strain is larger than 5mm, the strain is regarded as having cellulose degradation capacity and is marked as +; if the strain produces hydrolysis circles with a diameter of more than 3cm, the strain is considered to have stronger cellulose degradation capacity, which is marked as++.
2.2.2 measurement of carboxymethyl cellulase (CMCase) Activity
Inoculating the strain subjected to primary screening into TSB culture medium to obtain strain with concentration of about 10 8 Seed bacterial suspension of CFU is inoculated into a triangular flask of a rescreening culture medium with CMC-Na as a carbon source according to an inoculum size of 10% (v/v), shake culture is carried out at a constant temperature of 50 ℃, and the activities of carboxymethyl cellulose (CMCase) of 3, 4, 5, 6 and 7d strains are respectively measured, and 3 repeats are set.
(1) Drawing of a Standard Curve
The anhydrous glucose is dried to constant weight at 80 ℃ to prepare 1mg/mL standard glucose solution, 6 test tubes are taken, 0,0.2,0.4,0.6,0.8 and 1.0mL of standard glucose solution are respectively added, distilled water is added to 2.0mL, 1.5mL of DNS reagent is added, boiling water bath is carried out for 5min, the volume is fixed to 25mL after cooling, OD value is measured under spectrophotometry of 540nm, and a standard curve is drawn.
(2) Preparation of crude enzyme solution
Preparing a CMC-Na re-screening culture medium with a carbon source, subpackaging the culture medium into 250mL triangular flasks, 45mL each flask, inoculating 5mL of seed bacterial suspension, culturing in a shaking table at 28 ℃, respectively taking 1.5mL of fermentation liquor into a centrifuge tube after 7d of culture, and centrifuging for 10min at 10000r/min to obtain crude enzyme liquid.
(3) Enzyme Activity measurement
The method for measuring the enzyme activity comprises the following steps: 0.1mL of the crude enzyme solution was taken and 1.9mL of CMC-Na solution with a mass fraction of 1% was added. Hydrolyzing at 45deg.C for 20min, adding 1.5mL of DNS color development solution, performing boiling water bath for 5min, determining volume to 25mL, comparing color at 540nm, measuring absorbance (OD) value, comparing with standard glucose curve, and calculating glucose amount (m 1) from OD value. And (3) taking 0.1mL of crude enzyme solution, adding 1.9mL of water, adding 1.5mL of DNS color development solution, carrying out boiling water bath for 5min, carrying out constant volume to 25mL and carrying out color comparison at 540nm, and measuring the glucose amount (m 2) of the crude enzyme solution. Subtracting the glucose amount (m 2) from the glucose amount (m 1) to obtain the glucose amount truly obtained by degrading the CMC solution by CMC enzyme, calculating the glucose amount A by the optical density value, and calculating the enzyme activity (unit: mu mol/mL) of the cellulolytic bacteria under the above conditions by a formula: enzyme activity = a x 10 x 1000/20.
2.3 determination of protein degradation Capacity
2.3.1 preliminary screening for protein degradation Capacity
Uniformly coating the strain on a TSA culture medium, placing the TSA culture medium in a biochemical incubator at 50 ℃ for culture, observing that the strain grows uniformly on the surface of the culture medium, punching the uniformly grown strain together with the culture medium by a puncher with the diameter of 5mm to prepare a bacterial cake, inoculating the bacterial cake on a skimmed milk solid culture medium, placing the skimmed milk solid culture medium in the biochemical incubator at 50 ℃ for 2-4 days, observing whether a transparent hydrolysis ring is generated at the bacteria-inoculating position of the culture medium, measuring the diameter of the hydrolysis ring, and setting 3 repeats.
If the strain can not generate hydrolysis circles on the skimmed milk culture medium and the diameter of the generated hydrolysis circles is not more than 5mm, the strain is regarded as having no protein degradation capability and is marked as-; if the diameter of the hydrolysis circle generated by the strain is larger than 5mm, the strain is regarded as having protein degradation capability and is marked as +; if the strain generates hydrolysis circles with the diameter larger than 3cm, the strain is considered to have strong protein degradation capacity, and is marked as++.
2.3.2 measurement of protease Activity of Strain
(1) Drawing of a Standard Curve
L-tyrosine standard solution was prepared and measured immediately after dilution to 100. Mu.g/mL. Taking 1mL of diluted solution respectively, adding 5mL of 0.4mol/L sodium carbonate solution and 1mL of 1mol/L Folin-phenol reagent solution respectively, oscillating uniformly, developing in a water bath at 40 ℃ for 20min, measuring the absorbance at 680nm by a spectrophotometer respectively, taking absorbance A as an ordinate and tyrosine concentration as an abscissa, drawing a standard curve, and calculating the tyrosine amount (mug) when the absorbance is 1 by using a regression equation, wherein the K value is the absorbance constant K value, and the K value is 95-100.
(2) Preparation of crude enzyme solution
1 loop of pure culture stored on the inclined plane is selected and inoculated to protease seed culture medium, and is cultured for 12 hours at 50 ℃ in a shaking table 150rmp, and is inoculated to protease basic fermentation culture medium according to 1% (v/v), and after 48 hours of shaking table 150rmp culture at 50 ℃, the supernatant is centrifugally taken for enzyme activity measurement.
(3) Enzyme Activity measurement
And (3) carrying out enzyme activity measurement on the crude enzyme solution by adopting a Folin-phenol method. Definition of enzyme activity unit: hydrolysis of the casein substrate at 40℃per minute yields 1. Mu.g of tyrosine, defined as a unit of protease activity. Placing the casein solution with the mass fraction of 2% into a constant-temperature water bath with the temperature of 40 ℃ for preheating for 5min; adding 1mL of crude enzyme solution into 1mL of preheated casein solution, mixing, placing into 40 ℃ water bath, preserving heat for 10min, adding 2mL of 0.4mol/L trichloroacetic acid to terminate reaction, adding 0.4mol/L Na into 1mL of supernatant 2 CO 3 5mL, 1mL of Folin-phenol reagent was added thereto, and after 20min of color development in a water bath at 40℃the absorbance was measured at 660nm, and the reaction system with water was used as a blank.
Calculating the enzyme activity: and reading the enzyme activity of the final diluent from the standard curve, wherein the unit U/mL of the enzyme activity of the stock solution is calculated according to the following formula: protease activity (U/mL) =a×k×4 ≡10×n.
Wherein: a: OD values of parallel experiments of fermentation stock solutions; k: a light absorption constant; n: dilution factor of protease solution; 4: the total volume of the reactants; 10: the reaction time is 10min; the result is expressed as an integer.
2.4 determination of fat degradation Capacity
2.4.1 Primary screening for fat degradation Capacity
Victoria blue B is used as an indicator and turns blue when meeting acid. The lipase producing strain can produce lipase, and the lipase can decompose olive oil added into a culture medium to convert the olive oil into fatty acid, and Victoria blue B turns blue when meeting acid. The lipase producing ability can be judged according to the size of the color-changing ring.
Uniformly coating the strain on a TSA culture medium, placing the TSA culture medium in a biochemical incubator at 50 ℃ for culture, observing that the strain grows uniformly on the surface of the culture medium, punching the uniformly grown strain together with the culture medium by a puncher with the diameter of 5mm to prepare a bacterial cake, inoculating the bacterial cake on a Victoria blue B solid culture medium, placing the Victoria blue B solid culture medium in the biochemical incubator at 50 ℃ for 2-4 days, observing whether blue is generated at the strain inoculating position of the culture medium, measuring the diameter of a color-changing ring, and setting 3 repetitions.
If the strain cannot generate color-changing rings on the Victoria blue B culture medium and the diameter of the color-changing rings is not more than 5mm, the strain is regarded as not having fat degradation capability and is marked as-; if the diameter of the color-changing ring generated by the strain is larger than 5mm, the strain is regarded as having the capacity of degrading fat and is marked as +; if the diameter of the color-changing ring generated by the strain is larger than 3cm, the strain is considered to have stronger fat degradation capacity and is marked as++.
2.4.2 measurement of Lipase Activity of Strain
Several 100ml Erlenmeyer flasks were used, one as control flask and the other as assay flask, with the specific methods shown in Table 1.
TABLE 1 Lipase Activity determination method
Titration to reddish with 0.05M standard sodium hydroxide solution was performed and the volume of NaOH removed for titration was recorded.
And (3) calculating: specific lipase activity was defined as the amount of enzyme that hydrolyzes fat at pH 7.5 and 40℃to produce 1. Mu. Mol fatty acid per minute by 1g of lipase as one unit of enzyme activity.
Wherein: a is alkali liquor (m 1) consumed by the sample; b is alkali consumption liquid (m 1) of a control group; n is the concentration of the alkali liquor, namely 0.05 mu mol; f is the final dilution multiple of the crude enzyme solution; t is the action time (min).
2.5 experimental results
As a result of measurement, the temperature growth range of the bacterium Brevibacillus aydinogluensis PMBT001 was 40-70℃and the ability to produce cellulase, protease and lipase, and the results are shown in FIG. 3 and Table 2, in which the relative enzyme activity of cellulose was 21.7. Mu. Mol/mL, the relative enzyme activity of protease was 0.26. Mu. Mol/mL and the relative enzyme activity of lipase was 10.00. Mu. Mol/mL. The result shows that the high-temperature bacterial strain PMBT001 has better high-temperature resistant and enzyme producing capability and has the potential of further development and application.
TABLE 2 enzyme production ability of the strain PMBT001
Experimental example 3 application of bacterial Strain PMBT001 in cow dung
3.1 composting materials and Processes
Fresh cow dung comes from a large national farm in Zhaojuan county, sichuan province, china. The fresh manure is uniformly mixed with the auxiliary materials of urea, wood chips and wood shavings to form mixed manure, the mixed manure finally contains urea with the mass fraction of 0.2 percent, wood chips with the mass fraction of 25 percent and wood shavings, the mixed manure is randomly divided into 3 groups, the first group is not added with any microbial inoculant as a blank control group (CK), the second group is added with the microbial inoculant prepared in the example 1 (PMBT 001 group) according to the volume of 0.5 percent, and the third group is added with the microbial inoculant containing bacterial strain Bacillus sp Y3 according to the volume of 0.5 percent (Y3 group). Reference Zeng Min Bacillus diversity and copper tolerance studies [ J ]. Environmental sciences report, 2023, 43 (2): 485-495. The mixed manure is fermented for 14 days by adopting a stacking method in a manure treatment plant. Monitoring 8 a.m. using an automatic thermometer: compost daily temperature (Tm) changes of 00 and 6:00 PM, and compost around the thermometer (triplicate/day) was collected on days 1, 3, 5, 7, 9, 11, 13, 14, and analyzed for physicochemical properties and microbial characteristics. Thoroughly mixing the compost twice in the whole composting process, and turning the compost for the first time when the stacking temperature reaches more than 50 ℃; when the temperature is up to 60 ℃, the second turning is carried out to maintain the aerobic condition and uniformity.
3.2 physicochemical Property analysis
According to Chinese organic fertilizer agriculture rowIndustry Standard (NY/T525-2021) for pH, organic Matter (OM), total nitrogen (N), total phosphorus (P) of compost samples 2 O 5 ) Total potassium (K) 2 O) and seed Germination Index (GI). The pH of the 1:5 aqueous solution was measured with a pH meter (INESA PHSJ-3F, china). The organic matter content in the compost sample is determined by adopting a potassium dichromate titration method. Air-dried manure 0.25g in concentrated H 2 SO 4 And H 2 O 2 Middle digestion, determination of Total N, P 2 O 5 And K 2 O. Determining total nitrogen by Kjeldahl method, and determining total P by molybdenum blue colorimetric method 2 O 5 . Determination of total K by flame photometry 2 O. All experiments were performed 3 times.
3.3 high throughput sequencing analysis
3.3.1 16S rRNA V3-V4 sequencing procedure
0.25-0.3g of compost sample, extracting DNA by adopting a fecal genome DNA extraction kit (Tiangen Biochemical technology (Beijing) limited company), and detecting the purity and concentration of the DNA by adopting an ultra-micro ultraviolet spectrophotometer; taking 30ng of qualified genome DNA sample and corresponding fusion primer
338F:5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO. 4) and
806R:5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO. 5) is prepared into a PCR reaction system, PCR reaction parameters are set for PCR amplification, agencourt AMPure XP magnetic beads are used for purifying PCR amplification products and dissolving the PCR amplification products in an absorption Buffer, and labeling is carried out to complete library construction. Fragment ranges and concentrations of the library were detected using an Agilent 2100 Bioanalyzer. The library that was qualified was sequenced by selecting the HiSeq platform based on insert size.
3.3.2 high throughput sequencing information analysis procedure
Off-machine data are filtered, and the remaining high-quality Clean data are used for later analysis; splicing reads into Tags through an overlap relation between the reads; clustering Tags into OTUs and comparing with a database and annotating species; sample species complexity analysis, inter-group species difference analysis, correlation analysis, model prediction and the like are performed based on the OTU and the annotation result.
3.4 results
3.4.1 physicochemical results
(1) As shown in fig. 4, the temperature change of the compost sample in the composting process is shown in fig. 4, the temperature rise of the compost in the experimental group (PMBT 001 group) is faster, the compost rapidly enters the thermophilic phase and keeps stable on the 3 rd day after the start of the manure composting, the compost reaches the thermophilic phase faster than the CK (sterile group) and the experimental group (Y3 group), and the compost can maintain a longer high-temperature phase. The results demonstrate that in this study, the addition of a PMBT001 bacterial formulation to aerobic composting can accelerate the fecal composting into thermophilic phase and maintain the composting temperature.
(2) As shown in FIG. 5, the C/N change of the compost sample in the composting process is shown in FIG. 5, and as can be seen from FIG. 5, the organic matters are generally humified or mineralized by microorganisms in the composting process, and the carbon is accelerated to CO 2 Loss of form. Thus, changes in the C/N ratio in the compost sample reflect microbial activity in the heap. As shown in fig. 5, the C/N in the two stacks continued to drop, with CK, Y3 and PMBT001 groups dropping from 20.90, 22.76 and 20.19 to 17.08, 19.70 and 12.46, respectively, by 3.82, 3.06 and 7.73 orders of magnitude, respectively. This result is due to the strong mineralization of the carbonaceous compounds by the microorganisms in the thermophilic phase. Notably, the C/N in the PMBT001 group changed more significantly within 14d than the CK group, indicating that microbial activity was more active in the PMBT001 group.
(3) As shown in FIG. 6, the change of germination percentage (GI) of the compost sample seeds during the composting process is shown in FIG. 6, and the GI is a utilization index reflecting the maturity of the compost. The results showed that the GI of CK group was continuously decreased throughout the composting process from 79.41% on day 1 to 34.46% on day 14 (fig. 6), indicating that the accumulation of harmful substances during composting inhibited seed germination. The GI of the Y3 group increased from day 7 to day 9, but generally decreased from 71.29% to 37.04% after the composting period ended. The trend of decreasing and increasing GI of PMBT001 indicates that the harmful substances in the compost pile are removed after the composting period is ended. On day 14, PMBT001 (98.63) had GI greater than 80%, indicating compost maturity. This result also suggests that PMBT001 can accelerate the fecal composting process.
(4) As shown in FIG. 7, the pH change of the compost sample in the composting process is shown in FIG. 7, the pH value of the CK group is 7.34-7.67, the pH value of the Y3 group is 7.52-7.76, the pH value of the PMBT001 group is 7.21-7.42, the pH values of the CK group and the Y3 group are slightly higher than the pH value of the PMBT001 group, but the pH change of the three groups is not obvious.
(5) The total phosphorus and potassium changes of the composting samples in the composting process are shown in figures 8 and 9, and the total nutrients (total phosphorus and total potassium) are gradually increased along with the composting process according to figures 8 and 9, so that the nutrient content in the composting samples of the PMBT001 group is richer. The total phosphorus and total potassium of the PMBT001 group were significantly higher than those of the CK and Y3 groups on day 14.
3.4.2 high throughput sequencing results
(1) Overall flora change: the dynamic change of bacterial communities in the composting process is analyzed by adopting a high-throughput sequencing method. As shown in fig. 10, the CK group, Y3 group, and PMBT001 group obtained 2671, 2508, and 2509 OTUs, respectively, in total. PLS-DA analysis based on OTU data shows that the microbial community is greatly changed in the composting process; the CK group and the bacterial addition group are separated, which shows that bacterial addition has a great influence on the change of flora in the fermentation process.
The four dominant bacteria in each sample were Proteus (Proteus), bacteroides (Bacteroides), firmides (Firmides) and Proteus (Spirochaeta) at the initial stage of composting, respectively. The relative abundance of the Proteus in both groups decreased and that of the Thick-walled bacteria increased much. The relative abundance of the firmicutes in the PMBT001 group increased sharply to 68.39% on day 7 of composting and decreased to 36.69% on day 14, while the relative abundance of the firmicutes in the CK group increased slowly throughout composting (CK: 16.68-42.24%), and the relative abundance of the firmicutes in the Y3 group increased continuously throughout composting (Y3: 6.56-54.49%). The relative abundance of bacteroides in CK and Y3 groups decreased continuously, and PMBT001 group decreased and then increased.
During composting, microbiota fall on the genus level and vary greatly, most genera fall in abundance. The dominant bacteria at the initial stage were Pseudomonas, acinetobacter, petrimonas, sphaerocheta, bacteroides and Arcobacter. As the composting process proceeds, the relative abundance of Acinetobacter, petrimonas, sphaerocheta, bacteroides and arcobactor decreases substantially.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A plantBrevibacillus aydinogluensisPMBT001, having deposit No. GDMCC No:63294; the collection unit is the department of microbiology of the academy of sciences of Guangdong (collection of microbiological bacterial strains of Guangdong), the collection unit address: the Guangdong province, guangzhou City, first, china, no. 100, no. 59, 5, and Guangdong province microbiological institute, with a storage date of 2022, 03 and 27.
2. The method according to claim 1Brevibacillus aydinogluensisThe PMBT001 is characterized in that the 16S rRNA gene sequence is shown in SEQ ID NO. 1.
3. Claim 1 or 2Brevibacillus aydinogluensisApplication of PMBT001 in preparing microbial agent.
4. A microbial agent is characterized in that: comprising the composition according to claim 1 or 2Brevibacillus aydinogluensis PMBT001。
5. The microbial agent of claim 4, wherein: the saidBrevibacillus aydinogluensisThe number of viable bacteria of PMBT001 in the microbial agent is 1.0x10 8 -5.0×10 8 CFU/g。
6. The microbial agent of claim 4, wherein: the saidBrevibacillus aydinogluensis PMBT001 in the microThe number of viable bacteria in the biological bacterial agent is 1.3X10 8 CFU/g, pH value is 6.0-8.0.
7. The method for preparing the microbial agent according to any one of claims 4 to 6, characterized by comprising the steps of:
will beBrevibacillus aydinogluensis PMBT001 is inoculated in a TSB culture medium and cultured for 1 day at 50-60 ℃ and 160-200r/min to obtain seed liquid; inoculating the seed solution into a fermentation culture medium according to an inoculum size of 5% (v/v), and performing shake culture at 50-60 ℃ for 1-2 days at 160-200r/min to obtain a microbial agent;
the components of the fermentation medium are as follows: 5-10g of peptone, 1g of beef extract, 5g of sodium chloride, 10g of glucose and 1000mL of distilled water.
8. Claim 1 or 2Brevibacillus aydinogluensis Use of PMBT001 or a microbial agent according to any one of claims 4 to 5 for the preparation of a microbial fertilizer.
9. A microbial fertilizer, which is characterized in that: comprising the composition according to claim 1 or 2Brevibacillus aydinogluensis PMBT001 or the microbial agent of any one of claims 4-5.
10. Claim 1 or 2Brevibacillus aydinogluensis Use of PMBT001 or a microbial agent according to any one of claims 4 to 5 or a microbial fertilizer according to claim 9 for the high temperature aerobic composting of manure.
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| CN113507835A (en) * | 2018-11-15 | 2021-10-15 | 拜耳作物科学有限合伙公司 | Endospore display platform, product and method |
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| WO2021132036A1 (en) * | 2019-12-24 | 2021-07-01 | 住友化学株式会社 | Bacterium for degrading sludge, bacterium degrading microorganism, microbial preparation and method and device for degrading sludge |
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| Brevibacillus aydinogluensis sp. nov., a moderately thermophilic bacterium isolated from Karakoc hot spring;Kadriye Inan等;nternational Journal of Systematic and Evolutionary Microbiology;第62卷(第4期);第849–855页 * |
| 嗜热艾登短芽孢杆菌对聚乙烯醇的降解性能及机制;陈泽伟;湖南生态科学学报;第9卷(第2期);第1-9页 * |
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