CN112029690A - Bacillus solitarius and screening culture method and application thereof - Google Patents
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Abstract
The invention provides a strain of Bacillus sonoralis and a screening culture method and application thereof. The Bacillus sonoralis desert Bacillus is classified and named as Bacillus sonorensis CSB-5, is preserved in China center for type culture Collection in 5-11 months in 2020, and has the preservation number of CCTCC NO: m2020113. The screening culture method comprises the following steps: collecting a sample; coating the diluent of the sample on a nitrobacteria solid culture medium by adopting a gradient dilution method, and culturing at a constant temperature of 20-55 ℃; after the culture medium grows out of the colonies, selecting the colonies with different forms to perform streak culture on a beef extract peptone flat plate; subsequently, a loop of colonies was picked for repeated streaking with microscopic observation until a purified strain was obtained. The bacillus sonolatus can degrade and remove organic matters and ammonia nitrogen in domestic wastewater, breeding wastewater and industrial wastewater.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, in particular to a bacillus sonila desert and a screening culture method and application thereof.
Background
With the further advance of the work of the toilet revolution, a large number of parks, tourist areas and outdoor work areas are equipped with environment-friendly toilets. In three types of ecological toilets, namely, a packaging type ecological toilet, a water-free type ecological toilet and a circulating water type ecological toilet, the circulating water type microbial degradation toilet can realize the recycling of water resources and the reduction of excrement, has the advantages of safety, environmental protection, no odor and the like, and occupies a large market share at present.
In order to realize the water circulation of the circulating water flushing environment-friendly toilet, COD and ammonia nitrogen in waste water generated in the toilet need to be quickly removed, wherein, the biological denitrification technology is largely adopted due to the simplicity, high efficiency and low cost. The heterotrophic nitrifier can degrade the organic substrate in an aerobic environment and simultaneously carry out nitrification and denitrification to degrade ammonia nitrogen, thereby breaking through the view that the traditional denitrification can only be carried out under anaerobic conditions. Aerobic denitrification makes the construction of a synchronous denitrification system possible, so heterotrophic nitrification-aerobic denitrification bacteria become the key research objects. In recent years, research on heterotrophic nitrification-aerobic denitrification bacteria at home and abroad has been made with certain results, and found out bacteria capable of heterotrophic nitrification-aerobic denitrification include Ochrobactrum (Ochrobactrum), Pseudomonas (Pseudomonas), Parathiococcus (Paracoccus), Bacillus (Bacillus), Acinetobacter (Acinetobacter), Providencia (Providencia) and Rhodococcus pyridinivorans (Rhodococcus), and the above heterotrophic nitrification-aerobic denitrification bacteria all have wide application values in sewage treatment.
Therefore, more and better strains with the functions of heterotrophic nitrification-aerobic denitrification are screened, and the method has important significance for improving the denitrification efficiency of the wastewater.
Disclosure of Invention
The present invention aims to address at least one of the above-mentioned deficiencies of the prior art. For example, one of the purposes of the invention is to provide a strain of bacillus sonolatus capable of degrading and removing organic matters and ammonia nitrogen in domestic wastewater, and a screening culture method and application thereof.
In order to achieve the above object, one aspect of the present invention provides a strain of Bacillus sonoralis, which is classified and named as Bacillus sonoralis CSB-5, and is deposited in the chinese typical culture collection center at 5/11/2020 with a deposition number of CCTCC NO: m2020113.
In an exemplary embodiment of a strain of bacillus sonolatus of the present invention, the bacillus sonolatus is a gram-positive bacterium, has spores, is rod-shaped, is motile, and is obligatorily aerobic.
In an exemplary embodiment of a strain of bacillus sonolatus of the present invention, the 16S rDNA sequence of the bacillus sonolatus is shown in SEQ ID No. 1.
In an exemplary embodiment of the strain of Bacillus somnophilus, a bacterial colony formed after the Bacillus somnophilus is cultured on a beef extract peptone medium for 24 hours is circular or irregular, and a bacterial colony after 48 hours is circular and white, has a diameter of 0.5-0.8 mm, is neat in edge, and is flat and moist.
In an exemplary embodiment of a strain of bacillus sonolatus of the present invention, the bacillus sonolatus has stress resistance properties and can grow in a medium with a salt concentration of 0.5% to 8%.
The invention also provides a screening culture method of the Bacillus somnophilus, which comprises the following steps: collecting samples and storing the samples at low temperature, wherein the samples comprise septic tank sludge, livestock and poultry manure, soil, water, sludge and organic fertilizer; coating the diluent of the sample on a nitrobacteria solid culture medium by adopting a gradient dilution method, and culturing at a constant temperature of 20-55 ℃; after the culture medium grows out of the colonies, selecting the colonies with different forms to perform streak culture on a beef extract peptone flat plate; subsequently, a loop of colonies was picked for repeated streaking with microscopic observation until a purified strain was obtained.
The invention further provides application of the bacillus sonoralis or the bacterial suspension thereof or the culture solution thereof or the fermentation product thereof in degrading and removing organic matters and ammonia nitrogen in domestic wastewater, aquaculture wastewater and industrial wastewater.
In still another aspect of the present invention, there is provided a biological agent, which may include the above-mentioned bacillus solitarius or its bacterial suspension or its culture solution or its fermentation product.
The invention further provides the application of the bacillus sonoralis or the biological agent in degrading and removing organic matters and ammonia nitrogen in domestic wastewater, breeding wastewater and industrial wastewater.
In another aspect, the invention provides the application of the bacillus sonoralis or the biological agent in an ecological toilet.
Compared with the prior art, the invention has the advantages and beneficial effects that: the Bacillus sonorensis CSB-5 can effectively degrade and remove organic matters and ammonia nitrogen in domestic wastewater, breeding wastewater and industrial wastewater; under pure culture conditions, after inoculation culture for 72 hours, the degradation rate of the Bacillus sonolatus on organic matters of domestic wastewater reaches over 70.42 percent, the removal rate of ammonia nitrogen reaches over 79.12 percent, and the treatment effect is obvious; the bacillus sonolatus has strong stress resistance, can be used as an excellent treatment bacterium for treating domestic wastewater, aquaculture wastewater and industrial wastewater, and has good application prospect.
Drawings
The Bacillus sonoralis of the invention is preserved, and the preservation unit comprises: china Center for Type Culture Collection (CCTCC), preservation address: in the Wuhan university school of Wuhan 299 in the Wuchang area of Wuhan city, Hubei province, the preservation date is as follows: and 5, 11 months in 2020, the preservation number is CCTCC NO: m2020113.
The above and other objects and features of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings, in which:
FIG. 1A shows the microscopic form of Bacillus Desmophilus sonnardus according to an exemplary embodiment of the present invention; FIG. 1B shows the colony morphology of Bacillus somnophilus on beef extract peptone medium;
FIG. 2 shows a phylogenetic diagram of the 16S rDNA sequence of Bacillus cereus according to an exemplary embodiment of the present invention.
Detailed Description
Hereinafter, the Bacillus sonoralis of the present invention and its screening culture method and application will be described in detail with reference to the accompanying drawings and exemplary embodiments.
The invention provides a strain of Bacillus sonoralis, which is classified and named as Bacillus sonoralis CSB-5, is preserved in China center for type culture Collection in 5 and 11 months in 2020, and has a preservation number of CCTCC NO: m2020113.
In this example, Bacillus sonorensis CSB-5 is a gram-positive bacterium, has spores, rod-like, motile, and obligatory aerobic.
In this example, the 16S rDNA fragment was amplified by extracting the total DNA of strain CSB-5, and the 16S sequence was shown in SEQ ID NO. 1. The measured 16S sequence was compared with the National Center for Biotechnology Information (NCBI) database, and CSB-5 was compared with the model strain Bacillus sonnerensis (Bacillus sonorensis) NRRL B-23154 in GenBankTThe similarity of (C) to (D) was the highest (99.8%), and it was thus confirmed that CSB-5 was classified as Bacillus sonorensis CSB-5.
In the embodiment, a bacterial colony formed after Bacillus sonoralis (Bacillus sonorensis) CSB-5 is cultured on a beef extract peptone medium for 24 hours is circular or irregular, and a bacterial colony formed after 48 hours is circular and white, has the diameter of 0.5-0.8 mm, is neat in edge and is flat and wet.
In this example, Bacillus sonorensis CSB-5 has stress resistance and can grow in a medium with a salt concentration of 0.5% to 8%.
The invention also provides a screening culture method of the Bacillus somnophilus, which comprises the following steps:
1) collecting samples and storing the samples at low temperature, wherein the samples comprise septic tank sludge, livestock and poultry manure, soil, water, sludge and organic fertilizer;
2) coating a predetermined amount (for example, 3-8 g) of the dilution liquid of the sample on a nitrobacteria solid culture medium by adopting a gradient dilution method, and culturing at a constant temperature of 20-55 ℃; here, the sample is suitably used in an appropriate amount, and the temperature for cultivation may be adjusted depending on the temperature range of the environment to which the target strain is applied, for example, 5g of the sample may be used, 50 ℃ may be used if the target strain is applied to a high temperature environment, and 25 ℃ may be used if the target strain is applied to a normal temperature environment.
3) After the culture medium grows out of the colonies, selecting the colonies with different forms to perform streak culture on a beef extract peptone flat plate;
4) subsequently (for example, after culturing for 16-18 h), a ring of colonies is picked for repeated streaking, and combined with microscopic observation until a purified strain is obtained, and the purified strain is inoculated into a test tube slant culture medium of a beef extract peptone culture medium for preservation.
The Bacillus sonorensis CSB-5 has strong degradation capability and can degrade and remove organic matters and ammonia nitrogen in domestic wastewater, breeding wastewater and industrial wastewater.
Preferably, the bacterial suspension of the Bacillus sonorantis (CSB-5), or the culture solution thereof, or the fermentation product thereof also has strong degradation capability, and can effectively degrade and remove organic matters and ammonia nitrogen in domestic wastewater, aquaculture wastewater and industrial wastewater.
In yet another aspect of the invention, a biological agent is provided. In an exemplary embodiment of the biological agent of the present invention, the biological agent may include the Bacillus sonorantis CSB-5 of the present invention or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof.
The biological agent of the invention also has strong degradation capability, and can effectively degrade and remove organic matters and ammonia nitrogen in domestic wastewater, breeding wastewater and industrial wastewater.
The Bacillus sonorensis CSB-5 or the biological agent has good degradation application potential and can be applied to ecological toilets.
In order that the above-described exemplary embodiments of the invention may be better understood, further description thereof with reference to specific examples is provided below.
Example 1
(1) Separation, purification and preservation of Bacillus sonorensis CSB-5 (called bacterial strain CSB-5 for short) in desert
Collected septic tank sludge samples are stored at low temperature and taken back to a laboratory. 5g of sample is taken and coated on a nitrifying bacteria solid culture medium plate by adopting a gradient dilution method, and the nitrifying bacteria solid culture medium plate is cultured at the constant temperature of 30 ℃. After the colonies grow out, colonies with different forms are selected from the plate and streaked on a beef extract peptone plate. And (5) after culturing for 16h, picking one ring for repeated streak culture, and combining microscopic observation until purification. And inoculating the purified strain into a test tube slant culture medium of a beef extract peptone culture medium for storage.
The composition of the nitrifying bacteria solid culture medium is as follows: (NH4)2SO40.5g/L, sodium succinate 5.62g/L, K2HPO4 0.25g/L，MgSO4·7H2O 0.13g/L，NaCl0.13g/L，FeSO4·7H2O 0.0025g/L，MnSO4 ·4H2O0.025 g/L, deionized water 1L, pH 7.0, 121 deg.C sterilizing for 20 min.
As shown in FIG. 1, the separated and purified strain CSB-5 of the present example was cultured on a beef extract peptone medium, and the colony was circular, white, 0.5-0.8 mm in diameter, neat in edge, and flat and moist after 48 hours of culture. Wherein FIG. 1A shows the colony morphology and FIG. 1B is a schematic representation of colonies after streaking on beef extract peptone plates.
(2) Amplification and phylogenetic analysis of 16S rDNA of strain CSB-5
Extracting total DNA of the strain, using the total DNA as a template, using 27F and 1492R as primers to amplify a 16S fragment, and using a Bio-RADMCyclerTM instrument for PCR reaction.
Reaction system (50 μ l): mu.l of 2 XPCRMix 25. mu.l each of primers 27F and 1492R (10. mu.M), 1. mu.l of DNA template, and made up to 50. mu.l with ultrapure water; the nucleotide sequences of primers 27F and 1492R are shown in SEQ ID No.2 and SEQ ID No. 3.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 54 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 30 times; final extension at 72 ℃ for 8 min.
The PCR amplification product was detected on 1.0% agarose gel electrophoresis and then sent to Shanghai Bioengineering Co., Ltd for sequence determination. The software DNA man6.0 was used to perform the calculation of gene sequence similarity. The sequencing result is shown in SEQ ID No.1 (sequence table).
The obtained sequence results are compared in a National Center for Biotechnology Information (NCBI) database, and the similarity between the 16S rDNA gene sequence of the CSB-5 and the model strain of Bacillus sonorensis NRRL B-23154T in the database is the highest and is 99.8 percent. Based on the NCBI comparison results, the model strain with the highest similarity was selected as the reference strain, and phylogenetic trees (FIG. 2) and bootstrap values (bootstrap)1000 were constructed using the Neighbor-joining method (Neighbor-joining) of MEGA 6.0 software.
Based on the above characteristics, the strain CSB-5 was identified as Bacillus sonorensis. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2020, 5 months and 11 days, with the preservation number of CCTCC NO: m2020113.
(3) Experiment of COD capability of bacterial strain CSB-5 in degrading and removing domestic wastewater
The CSB-5 is inoculated on an LB liquid culture medium for activation, when the OD600 of the bacterial suspension subjected to activation culture is 1.0, the bacterial suspension is inoculated into sterilized 100mL of domestic wastewater (collected from a drilling team circulating water flushing ecological toilet aerobic aeration tank), the temperature is 30 ℃, the rotational speed is 140rpm for shaking culture, and the culture is repeated for 3 times by taking no inoculation as a control.
The strain capacity for degrading COD of domestic wastewater is determined: the culture solution after 72h of culture in the above test was collected, centrifuged at 10000rpm for 5min, the supernatant was collected, and the COD content thereof was measured and recorded (T1), and the initial COD content of the treated solution was measured and recorded in the same manner (T2). The COD removal rate (%) of the domestic wastewater is (T2-T1) 100/T1.
The measurement result shows that after 72 hours of culture, the COD removal rate of the domestic wastewater treated by inoculating the CSB-5 strain reaches 87.56%.
(4) Experiment for capability of bacterial strain CSB-5 in degrading and removing ammonia nitrogen in domestic wastewater
Inoculating CSB-5 on an LB liquid culture medium for activation, and when the OD600 of a bacterial suspension subjected to activation culture is 1.0, inoculating 5% of the bacterial suspension to sterilized 100mL of domestic wastewater (collected from a drilling team circulating water flushing ecological toilet aerobic aeration tank), wherein the ammonia nitrogen concentration is 244.36mg/L, carrying out shaking culture at 30 ℃ and 140rpm, and repeating for 3 times by taking no inoculation as a control.
The capacity of bacterial strain degradation for removing ammonia nitrogen in domestic wastewater is determined: centrifuging the fermentation liquor, collecting the supernatant, and respectively measuring NH in the culture solution4 +－N、NO3 －－N、NO2 －-concentration of N and TN, and converted to nitrogen content, denoted C1; the nitrogen content of the control fermentation broth was determined in the same manner and was designated as C2. The ammonia nitrogen removal rate (%) of the domestic wastewater is (C2-C1) 100/C2.
The measurement result shows that the ammonia nitrogen removal rate of the domestic wastewater treated by inoculating the CSB-5 strain reaches 79.12 percent after the domestic wastewater is cultured for 72 hours.
(5) Resistance test of Strain CSB-5
And (3) salt tolerance determination: preparing a beef extract peptone culture medium, and adjusting the pH value to 7; adding NaC1 into basic beef extract peptone medium, and preparing culture medium with salt concentration of 0.5%, 1%, 2%, 4%, 8% and 10%. After sterilizing at 121 ℃ for 30min, adding the bacterial suspension to a newly configured culture medium according to 1% (v: v), repeating three times, placing the culture medium in a 30 ℃ constant temperature incubator, performing shake culture at the rotating speed of 150r/min for 48h, and measuring the number of bacteria by using an ultraviolet spectrophotometer under the wavelength lambda of 600.
Measuring the growth temperature range: preparing a beef extract peptone culture medium, adjusting the pH value to 7, sterilizing at 121 ℃ for 30min, adding bacterial suspension to the newly-prepared culture medium according to 1% (v: v), placing the inoculated culture medium in a constant-temperature incubator of 10 ℃, 20 ℃, 30 ℃, 40 ℃, 55 ℃ and 65 ℃ respectively, carrying out shake culture at the rotating speed of 150r/min for 48h, setting 3 times at each temperature, and measuring the number of bacteria by using an ultraviolet spectrophotometer under the wavelength lambda of 600.
The result shows that the Bacillus sonoralis CSB-5 has good stress resistance and stronger salt tolerance and can grow in a beef extract peptone culture medium containing 8 percent of NaCl; the growth temperature range is wide, and the growth can be carried out within the temperature range of 20-55 ℃.
(5) Application of strain CSB-5 in ecological toilet experiment
The CSB-5 strain is inoculated in an LB liquid culture medium, is subjected to shaking culture at 28 ℃, is cultured for 24 hours, and is inoculated to an aerobic aeration tank of a circulating water flush type ecological toilet of a drilling team according to 5 percent, wherein the use frequency of the toilet can reach 100 people times per day. Taking a water sample from the aerobic aeration tank every two weeks, and determining COD and ammonia nitrogen content of the water sample. The experimental period was 8 weeks with no inoculation treatment as a control.
The results show (table 1): COD and ammonia nitrogen of the aerobic aeration tank inoculated with the treatment bacteria CSB-5 are both obviously lower than those of the control; after 4 weeks of inoculation treatment, the COD and ammonia nitrogen in the liquid in the aeration tank are not increased any more and are continuously reduced, and compared with the control treatment, the reduction rate of the COD and the ammonia nitrogen is more than 70 percent, thereby showing good treatment application potential.
TABLE 1 treatment effect of bacterial strain CSB-5 on COD and ammonia nitrogen in aerobic aeration tank
In conclusion, the Bacillus sonorensis CSB-5 can effectively degrade and remove organic matters and ammonia nitrogen in domestic wastewater, aquaculture wastewater and industrial wastewater; under pure culture conditions, after inoculation culture for 72 hours, the degradation rate of the Bacillus sonolatus on organic matters of domestic wastewater reaches over 70.42 percent, the removal rate of ammonia nitrogen reaches over 79.12 percent, and the treatment effect is obvious; the Bacillus sonorensis CSB-5 has strong stress resistance, can be used as an excellent treatment bacterium for treating domestic wastewater, aquaculture wastewater and industrial wastewater, and has good application prospect.
Although the present invention has been described above in connection with the exemplary embodiments and the accompanying drawings, it will be apparent to those of ordinary skill in the art that various modifications may be made to the above-described embodiments without departing from the spirit and scope of the claims.
Sequence listing
<110> Chuanqing drilling engineering Co., Ltd, China oil and gas group Co., Ltd
<120> Bacillus solitarius and screening culture method and application thereof
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ccggggctaa taccggatgc ttgattgaac cgcatggttc aattattaaa gggggctttt 180
aacttcccct ttccgatgga cccccggcgc attaactaat tggtgaggga acggctcccc 240
aaggggacga tgcgtaaccg acctgaaagg gtgatcggcc accctgggac tgaaacacgg 300
cccaaactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360
aacaacgccg cgtgagtgat gaagggtttc ggatcgtaaa actctggtgg tagggaaaaa 420
caagtaccgg tccaataggg cggtaccttg acggtaccta accagaaagc cacggctaac 480
tacctgccag cagccgcggt aatacgtagg tggcaagcgt tgkccggaat tattgggcgt 540
aaagcgcgcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600
gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660
tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720
gacgctgagg cgcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780
gtaaacgatg agtgctaagt gttagagggt ttccgccctt tagtgctgca gcaaacgcat 840
taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900
ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960
ttgacatcct ctgacaaccc tagagatagg gcttcccctt cgggggcaga gtgacaggtg 1020
gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080
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Claims (10)
1. The strain of Bacillus sonoralis is characterized in that the Bacillus sonoralis is classified and named as Bacillus sonoralis CSB-5, is preserved in China Center for Type Culture Collection (CCTCC) in 5-11 months in 2020, and has a preservation number of CCTCC NO: m2020113.
2. The bacillus sonolatus according to claim 1, wherein the bacillus sonolatus is a gram-positive bacterium, has spores, is rod-shaped, is motile, and is obligatorily aerobic.
3. The Bacillus sonolatopsis according to claim 1, wherein the 16S rDNA sequence of the Bacillus sonolatopsis is shown in SEQ ID No. 1.
4. The Bacillus sonolatopsis according to claim 1, wherein the bacterial colony formed by culturing the Bacillus sonolatopsis on a beef extract peptone medium for 24 hours is circular or irregular, and the bacterial colony after 48 hours is circular and white, has a diameter of 0.5-0.8 mm, and is neat in edge and flat and moist.
5. The bacillus sonolatus according to claim 1, characterized in that it has stress-resistant properties and can grow in a medium with a salt concentration of 0.5% to 8%.
6. The screening culture method of Bacillus sonolania according to claim 1, wherein the screening culture method comprises the following steps:
collecting samples and storing the samples at low temperature, wherein the samples comprise septic tank sludge, livestock and poultry manure, soil, water, sludge and organic fertilizer;
coating the diluent of the sample on a nitrobacteria solid culture medium by adopting a gradient dilution method, and culturing at a constant temperature of 20-55 ℃;
after the culture medium grows out of the colonies, selecting the colonies with different forms to perform streak culture on a beef extract peptone flat plate;
subsequently, a loop of colonies was picked for repeated streaking with microscopic observation until a purified strain was obtained.
7. The use of Bacillus sonolatus or its bacterial suspension or its culture solution or its fermentation product of claim 1 for degrading and removing organic matter and ammonia nitrogen from domestic wastewater, aquaculture wastewater and industrial wastewater.
8. A biological agent comprising the bacillus sonolatus or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof according to claim 1.
9. Use of the bacillus sonolatus according to claim 1 or the biological agent according to claim 8 for degrading and removing organic matters and ammonia nitrogen from domestic wastewater, aquaculture wastewater and industrial wastewater.
10. Use of the bacillus sonolatus according to claim 1 or the biological agent according to claim 8 in an ecological toilet.
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