CN115141770A - Bacillus tequilensis H1 capable of degrading COD in livestock wastewater and application thereof - Google Patents
Bacillus tequilensis H1 capable of degrading COD in livestock wastewater and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus tequilensis H1 capable of degrading COD in livestock wastewater and application thereof, and relates to the technical field of livestock wastewater treatment. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 4 months and 15 days in 2022 with the preservation number of CGMCC (China general microbiological culture Collection center). The invention obtains a new strain through screening, the strain is identified as bacillus tequilensis (Bacillus equilenis) through 16S rDNA molecules, the strain has the performance of rapid growth and degradation of high-concentration COD in livestock and poultry wastewater, the strain can realize the resource utilization of the livestock and poultry wastewater, and the problems of high treatment difficulty and easy environmental pollution of the livestock and poultry wastewater with high-concentration COD are solved.
Description
Technical Field
The invention belongs to the technical field of livestock and poultry wastewater treatment, and particularly relates to bacillus tequilensis H1 capable of degrading COD (chemical oxygen demand) in livestock and poultry wastewater and application thereof.
Background
Along with the improvement of living conditions, the demand of people for meat and eggs of livestock and poultry is increased, the amount of excrement generated by the fed livestock and poultry is increased, and the livestock and poultry is not properly treated, so that the environmental pollution is caused. The concentrated discharged livestock and poultry manure far exceeds the purification capacity of soil and plants in the area, so that the serious environmental pollution is caused, and the ecological balance is destroyed.
The livestock and poultry wastewater belongs to high-ammonia nitrogen and high-COD concentration organic wastewater and has complex components. Livestock wastewater is the most difficult to treat among livestock and poultry breeding wastes, so the treatment of the livestock and poultry wastewater is the key to realize the resource treatment of the breeding wastes, and at present, a chemical method and a microbial degradation method are adopted for treating high-concentration COD in the livestock and poultry wastewater, and the microbial degradation method needs to realize the degradation and removal of the COD in the livestock and poultry wastewater by means of the metabolic activity of degrading strains. In order to solve the problem of high-concentration COD treatment difficulty in the livestock wastewater at present, a strain specially used for degrading the high-concentration COD in the livestock breeding wastewater is separated and screened out at present so as to realize the efficient degradation of the COD in the livestock wastewater.
Disclosure of Invention
In order to solve the problems that the treatment difficulty of high-concentration COD in the livestock wastewater is high, the environmental pollution is easy to cause, and the resource utilization of the livestock wastewater is not facilitated, the invention provides the Bacillus tequilensis H1 capable of degrading the COD in the livestock wastewater and the application thereof.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the Bacillus tequilensis H1 capable of degrading COD in livestock wastewater is obtained by separating the Bacillus tequilensis H1 from the livestock wastewater, and the strain is preserved in China general microbiological culture Collection center (CGMCC for short, address: no. 3 of Beijing university Shih 1, china academy of sciences microorganism research center, zip code 100101) in 4 months and 15 days of 2022 years in the south China Committee for preservation and management of microbial strains, and the preservation number is CGMCC NO.24679, and is identified as Bacillus (Bacillus) and presumed to be Bacillus tequilensis (Bacillus tequilensis).
Preferably, the bacillus tequilensis H1 capable of degrading COD in the livestock wastewater is purple after gram staining and is gram positive bacteria.
Preferably, the bacillus tequilensis H1 capable of degrading COD in the livestock wastewater is screened from the livestock wastewater.
Preferably, the method for separating and purifying the bacillus tequilensis H1 capable of degrading COD in the livestock wastewater comprises the following steps: (1) sucking l mL of livestock and poultry wastewater cultured by the strain, and respectively diluting the wastewater to 10 ℃ according to a gradient dilution method -5 、10 -6 、10 -7 Taking 100 mu L of each diluent, respectively coating the diluent on a Maishi solid culture medium, and culturing for 2 days at 37 ℃ in an aseptic environment; (2) picking out colonies with different forms obtained by coating, inoculating into Maillard fluid medium, and culturing for 1 day at 30 deg.C and 140 r/min; (3) sucking l mL of liquid solution cultured for one day in the step (2), and respectively diluting to 10% by a gradient dilution method -5 、10 -6 、10 -7 Taking 100 mu L of each dilution, respectively coating the dilution on a new solid medium plate for culturing, repeating the operation for many times until a single strain is separated and purified, and then storing in glycerol.
Preferably, the invention also provides application of the bacillus tequilensis H1 capable of degrading COD in the livestock wastewater with high COD concentration.
Optimally, the Bacillus tequilensis H1 is respectively inoculated into the livestock wastewater with high COD concentration of 6000mg/L, 8000mg/L, 10000mg/L and 12000mg/L, the degradation rate of COD in the livestock wastewater inoculated with the Bacillus tequilensis H1 and the degradation rate of COD in the livestock wastewater not inoculated with the Bacillus tequilensis H1 are respectively detected and analyzed after 48 hours, and the degradation rate of COD in the livestock wastewater inoculated with the Bacillus tequilensis H1 reaches over 90 percent and is higher than that in the livestock wastewater not inoculated with the Bacillus tequilensis H1.
The invention has the following beneficial effects:
1. the strain is identified as bacillus tequilensis (Bacillus equilenis) through 16S rDNA molecules, has the performance of rapid growth and degradation of high-concentration COD in livestock wastewater, can realize the resource utilization of the livestock wastewater, and solves the problems of high treatment difficulty and easy environmental pollution of the livestock wastewater with high-concentration COD.
2. The Bacillus tequilensis H1 has the advantages of simple culture conditions, low cost, high efficiency and no secondary pollution, can be used for efficiently treating organic wastewater with high ammonia nitrogen and high COD concentration after being subjected to amplification culture, and has good engineering application prospect.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a characteristic diagram of a colony of Bacillus tequilensis H1 according to the present invention.
FIG. 2 is a gram-stained colony of Bacillus tequilensis H1 according to the present invention.
FIG. 3 is a graph showing the growth of colonies during the culture of Bacillus tequilensis H1.
FIG. 4 is a degradation diagram of COD in livestock wastewater when Bacillus tequilensis H1 is applied.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 screening, isolation and purification of Bacillus tequilensis H1
1. Screening of Bacillus tequilensis H1
Absorb a certain amount of beasts and birds waste water in follow beasts and birds waste water tank, carry out the dyeing process through gram stain method, this fungus bacterial colony characteristic is shown in figure 1, and the bacterial colony is whole to be irregular shape, and light red has metallic luster, and the smooth slight arch in surface, gram stain be purple, for gram-positive fungus, this fungus dyeing characteristic is shown in figure 2, then places this preliminary colony of discerning in preliminary liquid medium and carries out earlier stage culture, and this preliminary liquid medium specifically is: 3g of beef extract, 10g of peptone, 5g of NaCl, 15g of agar and 1000ml of distilled water, and adjusting the pH value to 7.0-7.4, so that the strain is propagated in a large amount in the liquid culture medium, and subsequent separation and purification are facilitated.
2. Isolation and purification of Bacillus tequilensis H1
(1) Absorbing l mL of livestock and poultry wastewater cultured by the strain, and respectively diluting the wastewater to 10 percent according to a gradient dilution method -5 、10 -6 、10 -7 Taking 100 mu L of each diluent, respectively coating the diluent in a Maifang solid culture medium, and culturing for 2 days at 37 ℃ in an aseptic environment;
(2) Picking out colonies with different forms obtained by coating, inoculating into Maillard fluid medium, and culturing for 1 day at 30 deg.C and 140 r/min;
(3) Sucking l mL of liquid solution cultured for one day in the step (2), and respectively diluting to 10% by a gradient dilution method -5 、10 -6 、10 -7 Taking 100 mu L of each diluent, respectively coating the diluent on a new solid culture medium flat plate for culture, and repeating the operation for multiple times until a single strain is separated and purified;
(4) And (3) storing the single strain obtained after separation and purification in the steps (1) to (3) in glycerol for later use.
3. Preparation of Maifang solid culture medium
10g of glucose, 8.2g of anhydrous sodium acetate, 2.5g of yeast extract, 1.8g of potassium chloride and 18g of agar are accurately weighed into 1000mL of distilled water, and the prepared Maishi solid culture medium is kept at a natural pH value.
The strain, namely the Bacillus tequilensis H1 for screening, separation and purification is preserved in China general microbiological culture Collection center (CGMCC for short, the address: no. 3 of the Ministry of microbiological research institute of China, the Zen West Lu No. 3 of the Chaxingxi district, beijing City, china), 4 months and 15 days in 2022, and the preservation number is CGMCC NO.24679.
4. Colony growth analysis in process of separating, purifying and culturing Bacillus tequilensis H1
Sucking a certain amount of purified strains preserved in glycerol, placing the purified strains in a new Maillard liquid culture medium, wherein the component composition of the Maillard liquid culture medium is the same as that of a primary liquid culture medium in the screening process of the Bacillus tequilensis H1, culturing for 72 hours in the new Maillard liquid culture medium, and periodically detecting the concentration of bacterial colonies in the liquid culture medium, wherein the figure 3 shows the growth and reproduction conditions of the bacterial colonies in the whole process of bacterial colony culture within 72 hours, and as can be seen from the figure 3, the H1 bacteria are in an adaptation period within 0-10 hours, the bacterial strains are just inoculated into the culture solution, a certain time is needed to adapt to a new environment, the bacterial strains are active in metabolism, slow in division and small in bacterial number increase during the period; the culture solution is in a logarithmic proliferation stage for 10 to 54 hours, the nutrient substances in the culture solution are sufficient, the proliferation of the strains is proliferated in a logarithmic mode, the number of the strains is greatly increased, and a large amount of nutrient substances are consumed; the strain is in a stable stage for 54 to 73 hours, a large amount of nutrient substances are consumed by the strain in a logarithmic stage, so that the strain cannot be continuously propagated, the number of the strain is kept relatively stable, the newly increased number and the death number are almost equal, at the moment, the bacteria can be changed in morphology and physiology, and most of the anabolic products of some bacteria are generated in the period.
Example 2 identification analysis of Bacillus tequilensis H1
(1) Genus analysis of the strain
Through searching documents related to Bacillus (Bacillus) and specific Bacillus tequilensis (Bacillus tequilensis) in the prior art, the strain is identified as Bacillus after comparative analysis, and specifically identified as Bacillus tequilensis, and the invention names the strain as Bacillus tequilensis H1, 16S rDNA sequence in a ribosome database for comparative analysis as shown in table 1.
TABLE 1.16 comparative analysis of S rDNA sequences in ribosomal databases
(2) 16S rDNA sequencing results
After PCR amplification is carried out by taking a bacterial genome as a template, a PCR product of the separated strain Bacillus tequilensis H1 is subjected to agarose electrophoresis with the mass fraction of 1 percent, and a fluorescence band of about 1500bp appears by electrophoresis observation and detection at 150V and 100mA for 20min. The size of the 16S rDNA fragment is 1456bp by direct sequencing of PCR primers, and the specific sequence is as follows:
based on the sequence, analysis shows that the strain H1 belongs to the same branch as the Bacillus tequilensis, and the 16S rDNA sequence analysis result shows that the genetic relationship with the known strain Bacillus tequilensis is the closest, and the homology reaches 100%. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: no. 3 of West Lu No. 1 of Beijing university Ind. Region, kyoho, china academy of sciences microbial research institute strain preservation management center, zip code 100101) at 15 days 4.2022, and the preservation number is CGMCC NO.24679.
Example 3 application of Bacillus tequilensis H1
According to the growth characteristics of the strains, the bacteria liquid which grows and propagates for 10 hours (namely the bacteria liquid in logarithmic phase) can be respectively and centrifugally inoculated into the livestock wastewater with high COD concentration of 6000mg/L, 8000mg/L, 10000mg/L and 12000mg/L, the growth condition is good, after 48 hours, the degradation rate of COD in the livestock wastewater inoculated with the Bacillus tequilensis H1 and the degradation rate of COD in the livestock wastewater not inoculated with the Bacillus tequilensis H1 are respectively detected and analyzed, the degradation rate of COD in the livestock wastewater inoculated with the Bacillus tequilensis H1 reaches over 90 percent and is obviously higher than that in the livestock wastewater not inoculated with the Bacillus tequilensis H1, at the moment, the strain in logarithmic phase utilizes a large amount of COD in the livestock wastewater as a nutrient for growth and propagation, and then the degradation removal of the COD in the livestock wastewater with high concentration is realized, and the specific degradation contrast is shown in figure 4, and the strain is a good engineering bacterium for the livestock wastewater.
In the description herein, references to the description of "one embodiment," "an example," "a specific example," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Sequence listing
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<120> Bacillus tequilensis H1 capable of degrading COD in livestock wastewater and application thereof
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gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac agatgggagc 60
ttgctccctg atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac 120
tgggataact ccgggaaacc ggggctaata ccggatggtt gtttgaaccg catggttcaa 180
acataaaagg tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg 240
tgaggtaacg gctcaccaag gcaacgatgc gtagccgacc tgagagggtg atcggccaca 300
ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360
ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct 420
ctgttgttag ggaagaacaa gtaccgttcg aatagggcgg taccttgacg gtacctaacc 480
agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540
ccggaattat tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc 600
cggctcaacc ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg 660
aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga 720
ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 780
ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag 840
tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca 900
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg 1020
gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg 1140
tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200
gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta 1260
agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag 1320
ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taacctttta 1440
ggagccagcc gccgaa 1456
Claims (6)
1. The Bacillus tequilensis H1 capable of degrading COD in livestock wastewater is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) with the preservation number of CGMCC NO.24679 in 2022, 4 months and 15 days.
2. The bacillus tequilensis H1 capable of degrading COD in livestock and poultry wastewater according to claim 1, wherein said bacillus tequilensis H1 is purple by gram staining and is a gram positive bacterium.
3. The Bacillus tequilensis H1 capable of degrading COD in livestock and poultry wastewater according to claim 1, which is selected from livestock and poultry wastewater.
4. The method for separating and purifying Bacillus tequilensis H1 capable of degrading COD in livestock wastewater according to any one of claims 1 to 3, characterized by comprising the following steps:
SS01 absorbs 1mL of livestock and poultry wastewater cultured by the strain, and the wastewater is respectively diluted to 10 according to a gradient dilution method -5 、10 -6 、10 -7 Taking 100 mu L of each diluent, respectively coating the diluent in a Maifang solid culture medium, and culturing for 2 days at 37 ℃ in an aseptic environment;
SS02 picking out colonies with different forms obtained by coating, inoculating the colonies into a Maitake liquid culture medium, and culturing for 1 day under the conditions that the temperature of a shaking table is 30 ℃ and the rotating speed is 140 r/min;
SS03 sucks liquid solution of the lmL cultured for one day in the step SS02, and the liquid solution is respectively diluted to 10 degrees according to a gradient dilution method -5 、10 -6 、10 -7 Taking 100 mu L of each diluent, respectively coating the diluent on a new solid culture medium flat plate for culturing, and repeating the operation for many times until a single strain is separated and purified;
SS04 the single strain obtained after the separation and purification of the steps SS01-SS03 is preserved in glycerol.
5. The use of the Bacillus tequilensis H1 according to claim 1 for degrading COD in livestock and poultry wastewater with high COD concentration in livestock and poultry wastewater.
6. The application of the Bacillus tequilensis H1 capable of degrading COD in the livestock wastewater with high COD concentration according to claim 5, characterized in that the Bacillus tequilensis H1 is respectively inoculated into the livestock wastewater with high COD concentration of 6000mg/L, 8000mg/L, 10000mg/L and 12000mg/L, the degradation rate of COD in the livestock wastewater inoculated with the Bacillus tequilensis H1 and the degradation rate of COD in the livestock wastewater not inoculated with the Bacillus tequilensis H1 are respectively detected and analyzed after 48 hours, and the degradation rate of COD in the livestock wastewater inoculated with the Bacillus tequilensis H1 reaches more than 90% and is higher than the degradation rate of COD in the livestock wastewater not inoculated with the Bacillus tequilensis H1.
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| CN115806349A (en) * | 2022-12-15 | 2023-03-17 | 鹤山市新的生物制品有限公司 | Method for increasing dissolved oxygen content of aquaculture water and special culture medium |
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