CN115141770B - 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 tertiaryii H1 capable of degrading COD in livestock and poultry wastewater and application thereof, and relates to the technical field of livestock and poultry wastewater treatment. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO.24679 at 4 and 15 of 2022. The invention screens and obtains a new strain which is identified as bacillus tertageus (Bacillus tertageus) by 16S rDNA molecules, the strain has the performances of rapid growth and capability of degrading high-concentration COD in livestock and poultry wastewater, the strain can realize the recycling of the livestock and poultry wastewater, and the problems of high treatment difficulty of the livestock and poultry wastewater with high-concentration COD and easy environmental pollution are solved.
Description
Technical Field
The invention belongs to the technical field of livestock wastewater treatment, and particularly relates to bacillus tertagatose H1 capable of degrading COD (chemical oxygen demand) in livestock wastewater and application thereof.
Background
Along with the improvement of living conditions, the demand of people for livestock, poultry, meat and eggs is increased, the quantity of raised livestock and poultry is increased, so that the generated excrement is increased, and the livestock and poultry excrement is improperly treated, thereby causing environmental pollution. The amount of the livestock and poultry manure which is discharged in a concentrated way far exceeds the purifying capacity of soil and plants in the region, so that serious environmental pollution is caused, and ecological balance is destroyed.
The livestock and poultry wastewater belongs to organic wastewater with high ammonia nitrogen and high COD concentration, and has complex components. The livestock wastewater is the most difficult to treat in livestock and poultry breeding waste, so the treatment of the livestock and poultry wastewater is the key for realizing the recycling treatment of the breeding waste, the current treatment method aiming at high-concentration COD in the livestock and poultry wastewater comprises a chemical method and a microbial degradation method, 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 degradation strains. In order to solve the problem of high difficulty in treating high-concentration COD in livestock wastewater, a strain special for degrading high-concentration COD in livestock and poultry breeding wastewater is screened out by separation so as to realize efficient degradation of COD in livestock and poultry wastewater.
Disclosure of Invention
The bacillus tervelarium H1 capable of degrading COD in the livestock and poultry wastewater and the application thereof provided by the invention can be used for carrying out metabolic decomposition by taking high-concentration organic matters in the livestock and poultry wastewater as nutrients, so that the technical problem of high treatment difficulty of the livestock and poultry wastewater is solved to a certain extent.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the invention relates to a Bacillus tefrasis H1 capable of degrading COD in livestock and poultry wastewater, which is separated from the livestock and poultry wastewater, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC, address: north Xiyan Lu No. 1, no. 3 of Beijing, chaiyan Korea, beijing, and the microbiological institute of culture collection center, post code 100101) in 4 months and 15 days of 2022, and is identified as Bacillus and presumed to be Bacillus tequilensis (Bacillus tefrasis).
Preferably, the bacillus tertiaryanaerobacter H1 capable of degrading COD in livestock and poultry wastewater is dyed into purple by gram and is a gram positive bacterium.
Preferably, the bacillus tertiaryii H1 capable of degrading COD in the livestock wastewater is selected from the livestock wastewater.
Preferably, the separation and purification method of the bacillus tertiaryalis H1 capable of degrading COD in livestock and poultry wastewater comprises the following steps: (1) sucking l mL of livestock and poultry wastewater subjected to strain culture, and respectively diluting to 10 according to a gradient dilution method -5 、10 -6 、10 -7 Doubling, taking 100 mu L of each diluent, respectively coating the diluent in a Maillard solid culture medium, and culturing for 2 days at 37 ℃ in a sterile environment; (2) picking out the colonies with different forms obtained by coating, inoculating to a Ma's liquid culture medium, and culturing for 1 day under the conditions of a shaking table temperature of 30 ℃ and a rotating speed of 140 r/min; (3) sucking l mL of the liquid solution after the culture in the step (2) for one day, and respectively diluting to 10 according to a gradient dilution method -5 、10 -6 、10 -7 Multiple times, 100 μl of each dilution was applied to a new solid medium plate for culture, and the procedure was repeated multiple times until a single strain was isolated and purified, and then stored in glycerol.
Preferably, the invention also provides application of the bacillus tervelarius H1 capable of degrading COD in the livestock wastewater in high COD concentration livestock wastewater.
Optimally, the application is that the bacillus tertiaryii H1 is respectively inoculated into livestock and poultry wastewater with high COD concentration of 6000mg/L, 8000mg/L, 10000mg/L and 12000mg/L, and after 48 hours, the degradation rate of COD in the livestock and poultry wastewater inoculated with the bacillus tertiaryii H1 and the degradation rate of COD in the livestock and poultry wastewater not inoculated with the bacillus tertiaryii H1 are respectively detected and analyzed, and the degradation rate of COD in the livestock and poultry wastewater inoculated with the bacillus tertiaryii H1 reaches more than 90 percent and is higher than the degradation rate of COD in the livestock and poultry wastewater not inoculated with the bacillus tertiaryii H1.
The invention has the following beneficial effects:
1. the invention screens and obtains a new strain which is identified as bacillus tertageus (Bacillus tertageus) by 16S rDNA molecules, the strain has the performances of rapid growth and capability of degrading high-concentration COD in livestock and poultry wastewater, the strain can realize the recycling of the livestock and poultry wastewater, and the problems of high treatment difficulty of the livestock and poultry wastewater with high-concentration COD and easy environmental pollution are solved.
2. The bacillus tertiaryii H1 in the invention has simple culture conditions, low cost, high efficiency and no secondary pollution, can be used for efficiently treating high ammonia nitrogen and high COD concentration organic wastewater after the amplification culture, and has good engineering application prospect.
Of course, it is not necessary for any one product to practice the invention to achieve all of the advantages set forth above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing colony characteristics of Bacillus tertefralis H1 of the present invention.
FIG. 2 is a graph showing colonies of Bacillus tertakii H1 of the present invention after gram staining.
FIG. 3 is a graph showing the growth of colonies during the culture of Bacillus tequilensis H1.
FIG. 4 is a diagram showing the degradation of COD in livestock wastewater when Bacillus tequilensis H1 is used.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 screening, isolation and purification of Bacillus tertakii H1
1. Screening of Bacillus tequilensis H1
Absorbing a certain amount of livestock wastewater from a livestock wastewater storage tank, performing dyeing treatment by a gram dyeing method, wherein the bacterial colony is characterized in that the bacterial colony is shown in fig. 1, the whole bacterial colony is in an irregular shape and is light red, has metallic luster and smooth and slightly convex surface, the gram dyeing is purple and is gram positive bacteria, the bacterial dyeing characteristic is shown in fig. 2, and then placing the primarily identified bacterial colony in a primary liquid culture medium for pre-culture, wherein the primary liquid culture medium is specifically: 3g of beef extract, 10g of peptone, 5g of NaCl, 15g of agar, 1000ml of distilled water and pH of 7.0-7.4 are regulated, so that the strain is propagated in a large amount in the liquid culture medium, and the subsequent separation and purification are facilitated.
2. Separation and purification of Bacillus tefraensis H1
(1) Sucking l mL of livestock and poultry wastewater subjected to strain culture, and respectively diluting to 10 according to a gradient dilution method -5 、10 -6 、10 -7 Doubling, taking 100 mu L of each diluent, respectively coating the diluent in a Maillard solid culture medium, and culturing for 2 days at 37 ℃ in a sterile environment;
(2) Picking out the colonies with different forms obtained by coating, inoculating to a Ma's liquid culture medium, and culturing for 1 day under the conditions of a shaking table temperature of 30 ℃ and a rotating speed of 140 r/min;
(3) Sucking l mL of the liquid solution after the culture in the step (2) for one day, and respectively diluting to 10 according to a gradient dilution method -5 、10 -6 、10 -7 Doubling, taking 100 mu L of each diluent, respectively coating on a new solid culture medium 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 the separation and purification in the steps (1) - (3) in glycerol for later use.
3. Preparation of Mahalanobis solid Medium
Accurately weighing 10g of glucose, 8.2g of anhydrous sodium acetate, 2.5g of yeast extract, 1.8g of potassium chloride and 18g of agar in 1000mL of distilled water, and keeping the prepared Maillard solid culture medium at natural pH.
The strain Bacillus tequilensis H1 for screening, separation and purification is preserved in China general microbiological culture Collection center (CGMCC, address: north Chen Xila No. 1, 3 of the area of Chaoyang in Beijing, china academy of sciences microbiological culture Collection center, post code 100101) at 4 and 15 days of 2022, and the preservation number is CGMCC No.24679.
4. Colony growth analysis in bacillus tertefraxins H1 separation, purification and culture process
Absorbing a certain amount of purified bacterial strain stored in glycerol, placing the purified bacterial strain in a new Mahalanobis liquid culture medium, wherein the composition of the components of the Mahalanobis liquid culture medium is the same as that of the primary liquid culture medium in the screening process of bacillus tertiaryalis H1, culturing the bacterial strain in the new Mahalanobis liquid culture medium for 72 hours, and periodically detecting the concentration of the bacterial colony in the liquid culture medium, wherein the bacterial colony grows and grows in the whole process of culturing the bacterial colony within 72 hours, and the bacterial strain H1 is in an adaptation period within 0-10 hours, and the bacterial strain is just inoculated into a culture solution and needs a certain time to adapt to a new environment, so that the bacterial strain is active in metabolism, slow in division and the bacterial number is not increased greatly; the culture medium is in a logarithmic proliferation period for 10 to 54 hours, nutrient substances in the culture medium are sufficient, the proliferation of strains is carried out in a logarithmic mode, the number of the strains is greatly increased, and a large amount of nutrient substances are consumed; the 54-73 hours are in the stable period, the strains consume a large amount of nutrient substances in the logarithmic phase, so that the strains cannot continue to reproduce, the number of the strains is kept stable, the newly increased number and the death number are almost equal, at the moment, the bacteria can change in morphology and physiology, and anabolic products of some bacteria are mostly produced in the period.
EXAMPLE 2 identification assay of Bacillus tertakii H1
(1) Analysis of genus of the strain
The strain was identified as Bacillus, specifically Bacillus, after comparative analysis by looking up prior art literature on Bacillus and specifically Bacillus tertagatous (Bacillus tequilensis) and was designated Bacillus tertagatous by the present invention as Bacillus tertagatous H1, 16S rDNA sequence in the ribosome database comparative analysis as shown in Table 1.
TABLE 1.16 comparison of rDNA sequences in ribosomal database
(2) 16S rDNA sequencing results
After PCR amplification by taking a bacterial genome as a template, the PCR product of the isolated strain bacillus tebruensis H1 is subjected to agarose electrophoresis with the mass fraction of 1%, and the fluorescence band of about 1500bp is observed and detected by electrophoresis with the concentration of 150V and 100mA for 20 min. The 16S rDNA fragment is 1456bp in size by direct sequencing of PCR primers, and the specific sequence is as follows:
based on the analysis of the sequence, the strain H1 and the bacillus tertiarygensis belong to the same branch, and the 16S rDNA sequence analysis result shows that the genetic relationship with the known strain bacillus tertiarygensis is closest, and the homology reaches 100%. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, address: north Xila No. 1, 3 of the Korean area of Beijing, china center for type-A microbiological culture Collection center, mail code 100101) of 4 months and 15 days of 2022, and the preservation number is CGMCC No.24679.
EXAMPLE 3 use of Bacillus tertakii H1
According to the growth characteristics of the strain, bacterial solutions (i.e. bacterial solutions in logarithmic phase) which grow and reproduce for 10 hours can be respectively centrifugally inoculated into livestock and poultry wastewater with high COD concentration of 6000mg/L, 8000mg/L, 10000mg/L and 12000mg/L, the growth condition is good, the degradation rate of COD in the livestock and poultry wastewater inoculated with the Bacillus tequilegia H1 and the degradation rate of COD in the livestock and poultry wastewater not inoculated with the Bacillus tequilegia H1 are respectively detected and analyzed after 48 and H, the degradation rate of COD in the livestock and poultry wastewater inoculated with the Bacillus tequilegia H1 reaches more than 90%, the degradation rate of COD in the livestock and poultry wastewater not inoculated with the Bacillus tequilegia H1 is obviously higher than that of COD in the livestock and poultry wastewater not inoculated with the Bacillus tequilegia H1, and a large amount of COD in the logarithmic phase is used as the feed for growth and reproduction at the moment, so that the degradation and removal of the high-concentration COD in the livestock and poultry wastewater are realized, and the degradation contrast is shown in figure 4, and is a good engineering strain for the livestock and poultry wastewater.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, 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 present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. 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 only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form 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 understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Sequence listing
<110> Anyang institute of technology
<120> Bacillus tertakii H1 capable of degrading COD in livestock and poultry wastewater and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1456
<212> DNA
<213> Bacillus tertefraxins (Bacillus tequilensis)
<400> 1
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 (5)
1. Bacillus tefrasis H1 (Bacillus tequilensisH 1) capable of degrading COD in livestock and poultry wastewater is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 24679 in the year 2022 and the month 4 and 15.
2. The bacillus tertiaryii H1 capable of degrading COD in livestock wastewater of claim 1, wherein the bacillus tertiaryii H1 is gram-stained purple and is a gram-positive bacterium.
3. The bacillus tertiaryii H1 capable of degrading COD in livestock wastewater of claim 1 is selected from livestock wastewater.
4. The use of bacillus tertiaryii H1 capable of degrading COD in livestock wastewater according to claim 1 in high COD concentration livestock wastewater.
5. The application of the bacillus tertiaryii H1 capable of degrading COD in livestock and poultry wastewater in high COD concentration according to claim 4, wherein the bacillus tertiaryii H1 is respectively inoculated into the livestock and poultry wastewater with high COD concentration of 6000mg/L, 8000mg/L, 10000mg/L and 12000mg/L, and after 48 hours, the degradation rate of COD in the livestock and poultry wastewater inoculated with the bacillus tertiaryii H1 and the degradation rate of COD in the livestock and poultry wastewater not inoculated with the bacillus tertiaryii H1 are respectively detected and analyzed, and the degradation rate of COD in the livestock and poultry wastewater inoculated with the bacillus tertiaryii H1 reaches more than 90 percent and is higher than the degradation rate of COD in the livestock and poultry wastewater not inoculated with the bacillus tertiaryii H1.
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