CN112877267A - Tropical bacillus strain and application thereof - Google Patents
Tropical bacillus strain and application thereof Download PDFInfo
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- CN112877267A CN112877267A CN202110431127.9A CN202110431127A CN112877267A CN 112877267 A CN112877267 A CN 112877267A CN 202110431127 A CN202110431127 A CN 202110431127A CN 112877267 A CN112877267 A CN 112877267A
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
Abstract
The invention relates to the technical field of microbial remediation of environmental pollution, and particularly relates to a bacillus tropicalis strain and application thereof. The tropical Bacillus strain is tropical Bacillus ADOM11, which is preserved in China general microbiological culture Collection center at 25.1.25.2021.A preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, a preservation number is CGMCC No.21730, and the strain is classified and named as tropical Bacillus tropicus; can be used for remedying the organic pollution of the water bloom water body. The bacillus tropicalis ADOM11 can promote decomposition of dissolved organic matters in an algae source, reduce organic pollution of a water body from the source, facilitate conversion and utilization of nutrient components of the water body, increase nutrient competition between microorganisms and algae, correspondingly reduce total nutrient supply of the algae, and finally achieve the technical effects of reducing the water bloom degree and shortening the water bloom process.
Description
Technical Field
The invention relates to the technical field of microbial remediation of environmental pollution, in particular to a Bacillus tropicalis (Bacillus tropicus) strain and application thereof.
Background
In recent years, the situation that the eutrophication degree of environmental water is deepened due to the discharge of domestic sewage and culture water is more common, the outbreak of cyanobacterial bloom is frequent, algae cells are eliminated and released algae toxins cause the pollution of water and aquatic products, and finally the safety and health of human beings are threatened.
The dissolved organic matter is a soluble organic component released by decomposing solid phase particle organic matter such as residual bait, animal and plant bodies and the like, is widely existed in various water bodies, and plays a key role in the nutrient salt circulation and eutrophication process of an aquatic ecosystem. Through intensive research on the eutrophication phenomenon mechanism, people find that the eutrophication phenomenon is not only a problem of excessive nutrient salts such as nitrogen and phosphorus, but also can be strictly defined as an increasing process of the input rate of (dissolved) organic matters in an ecosystem.
In a water area where the bloom occurs, algae exist as organic matters in large quantity, and the cells of the algae die and decompose to generate a large quantity of self-growing sources (algae sources) which dissolve the organic matters and continuously migrate to be released into the water body, so that the organic pollution load of the water body is directly increased; the algae source dissolved organic matter with high solubility contains rich biogenic elements such as carbon, nitrogen, phosphorus and the like as a dissolved organic carbon storage, can be supplied to algae cells for rapid growth of the algae cells so as to promote the continuous development and succession of water bloom, and forms a cycle of dissolving the organic matter by the algae source. Due to the short growth cycle of algae, the dissolved organic matters in the algae source can be continuously accumulated along with the progress of the water bloom, and particularly, in the end stage of the water bloom, a large amount of the dissolved organic matters in the algae source can be released by massive death of algae cells, so that the deterioration of water quality is caused quickly. Based on the method, how to timely and effectively remove the algae source dissolved organic matters becomes the key of water bloom treatment, and the method has important significance in the water bloom prevention and control process.
Microorganisms are an important component of the ecosystem and have historically played an important role in environmental pollution remediation. The bacterial cells play an important role in the treatment of cyanobacterial bloom, for example, certain algicidal bacteria can digest the cell wall of a host by directly contacting the algae cells and secreting cellulase so as to dissolve the algae cells, and other most bacteria can achieve the aim of inhibiting algae by competing for nutrient substances such as nitrogen, phosphorus and the like. The algae source dissolved organic matter in the water body can be degraded by natural light and is decomposed mainly by bacteria, but the degradation speed and the effective degree of different bacteria are different, and not all heterotrophic bacteria can degrade the dissolved organic matter.
Disclosure of Invention
Aiming at the technical problem that the final stage of algal bloom is dead in a large amount and releases a large amount of algae source dissolved organic matters to cause deterioration of water quality, the invention provides a tropical bacillus strain and application thereof, wherein the tropical bacillus ADOM11 can promote the decomposition of the algae source dissolved organic matters, reduce organic pollution of a water body from the source, facilitate the conversion and utilization of nutrient components of the water body, increase the nutrient competition between microorganisms and algae and correspondingly reduce the total amount of nutrient supply of the algae, finally achieve the technical effects of reducing the degree of the algal bloom and shortening the history of the algal bloom, and has important practical significance and application prospect.
In a first aspect, the invention provides a tropical Bacillus strain, wherein the tropical Bacillus strain is tropical Bacillus ADOM11, which is preserved in China general microbiological culture Collection center at 25.1.1st in 2021, and the preservation address is No. 3 of West Lu No. 1 of the Korean area in Beijing, the preservation number is CGMCC No.21730, and the strain is classified and named as tropical Bacillus tropicus. The strain can obviously degrade algae sources to dissolve organic matters, and reduce the organic pollution degree of algae in water, thereby purifying the water.
In a second aspect, the invention provides an application of the bacillus tropicalis ADOM11 in repairing organic pollution of a water bloom water body, and particularly provides an application of bacillus tropicalis ADOM11 in degrading an algae source dissolved organic matter.
Further, bacillus tropicalis ADOM11 bacterial liquid is added into a water body containing algae source dissolved organic matters for degradation.
Further, the bacillus tropicalis ADOM11 bacterial liquid is obtained by inoculating bacillus tropicalis ADOM11 into a sterilized nutrient broth culture solution, shaking for culture, and centrifuging and suspending in physiological saline.
Furthermore, the viable count of the bacillus tropicalis ADOM11 bacterial liquid is 5 × 108-6×108one/mL.
Further, the bacillus tropicalis ADOM11 bacterial liquid is added into a water body containing algae source dissolved organic matters according to the volume ratio of 3-10% for degradation.
Furthermore, the degradation time is more than 12 h.
Furthermore, the viable count of the bacillus tropicalis ADOM11 bacterial liquid is 5.53 multiplied by 108-6×108Adding the strain per mL of the organic matter dissolved in the algae source into a water body containing the algae source dissolved organic matter according to the volume ratio of 5%, and degrading for more than 72 h.
The beneficial effect of the invention is that,
the bacillus tropicalis ADOM11 provided by the invention has strong capability of degrading algae source dissolved organic matters, the removal rate of the algae source dissolved organic matters reaches 91.0% within a certain time, the restoration degree of organic pollution of a water body reaches 82.9%, the water quality is greatly improved, and the ecological benefit is improved.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a phylogenetic tree of Bacillus tropicalis ADOM 11;
FIG. 2 is a UV-Vis spectral absorption curve of example 3;
FIG. 3 is a graph showing the change in the concentration of dissolved organic carbon in example 3;
FIG. 4 is a curve showing the change of chemical oxygen demand in example 3.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all 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.
The algae-derived organic matter-dissolving culture solution used in the following examples was prepared as follows:
the following table 1 shows the main algae composition of the sampled water. Centrifuging a water sample (5000r/min, 3min) to obtain algae slurry, washing the algae slurry with ultrapure water, adding 10g (wet weight) of the algae slurry into 50mL of ultrapure water, uniformly mixing, ultrasonically crushing by using an ultrasonic cell crusher, quickly freezing in liquid nitrogen, thawing, repeating for 2 times, breaking the algae cells, filtering the solution by using a 0.45-micrometer filter membrane, sterilizing by high pressure and damp heat (121 ℃, 20min) to obtain 10 times of algae source dissolved organic matter culture solution, and storing in a sealed manner for bacteria prevention, light protection and low temperature. When it is used, the stock solution is diluted to 1x working concentration with 0.9% physiological saline, adjusted to pH7.8, and subpackaged in conical flask for sterilization (121 deg.C, 20min) for use.
TABLE 1 Main algal composition of sampled Water
The nutrient broth culture medium is provided by Qingdao Gaokoubo biotechnology limited company, and the formula of the nutrient broth culture medium comprises 10g/L peptone, 3g/L beef extract powder and 5g/L sodium chloride, and the pH value is 7.2 +/-0.2 (25 ℃); the slant culture medium is solid nutrient broth culture medium, and is prepared by adding 2.0% agar powder into nutrient broth culture medium, sterilizing at 121 deg.C for 20min under high pressure, and pouring the slant.
EXAMPLE 1 enrichment, isolation, purification and screening of the strains
Taking a water body of a grass carp culture pond with water bloom as a degrading bacteria separation source, performing enrichment culture by using an algae source dissolved organic matter culture solution, then performing purification separation for multiple times by using a traditional coating flat plate and scribing separation method to finally obtain 19 bacteria, respectively placing each strain of bacteria in 100mL of nutrient broth culture medium, performing activation culture for 1-2 days at 28 ℃ and 250rpm, then taking out 5mL of bacteria, respectively adding the bacteria into 100mL of algae source dissolved organic matter culture solution, performing culture at 28 ℃, 250rpm and in a dark place, periodically sampling, inspecting and comparing degradation effects of each bacteria on algae source dissolved organic matters, finally screening out a strain with higher capacity of promoting the decomposition of the algae source dissolved organic matters, and numbering ADOM 11; inoculating to slant culture medium, storing at 4 deg.C, inoculating to seed-preserving tube, and storing at-80 deg.C.
Example 2 molecular characterization and biological characterization of 16S rDNA of strains
Performing PCR amplification on the 16S rDNA molecular fragment of the strain by using primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3'), performing agarose gel electrophoresis detection, and performing bidirectional sequencing to obtain the 16S rDNA sequence of the strain:
GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTC。
the sequencing sequence of ADOM11 was submitted to GenBank for alignment, and ADOM11 and Bacillus tropicus strain AOA-CPS1 were found to have 100% homology and were named as Bacillus tropicalis ADOM11, and the phylogenetic tree of this strain is shown in FIG. 1.
The strain is a good-culture gram-positive bacterium, rod-shaped, sports, spore-forming, and when the strain grows on a nutrient broth culture medium, the bacterial colony is approximately round, white, opaque, flat and rough in surface.
Example 3
Activating the preserved strain on slant culture medium to obtain strain suspension, inoculating into 100mL sterilized nutrient broth culture medium, shake culturing at 28 deg.C and 250rpm for 36 hr, introducing into exponential growth phase, and culturing with 5mL strain solution (5.53 × 10)8cell/mL) centrifugation (10000r/min, 30min) to obtain thallus cells, then re-suspending with 5mL sterilized normal saline, inoculating into 100mL sterilized algae source dissolved organic matter culture solution for culture, wherein the dissolved organic matter concentration (calculated by dissolved organic carbon) of the culture solution is about 77mg/L, and chemical oxygen demand CODMnThe value was 105mg/L and a blank control was set. The culture conditions were set as follows: at 28 ℃ and 250rpm, protected from light.
Filtering the culture solution every 12h with a 0.22 μm filter membrane, measuring the ultraviolet-visible spectrum absorption curve, the dissolved organic carbon content and the chemical oxygen demand value of the filtrate, and continuously monitoring for 72h, wherein the ultraviolet-visible spectrum absorption curve is scanned in a range of 230 plus 500nm (with 1cm quartz cuvette, interval of 1nm and pure water as reference) by using an ultraviolet-visible spectrophotometer; measuring dissolved organic carbon by using a TOC instrument; the chemical oxygen demand adopts an acid potassium permanganate method, a sample is diluted to a proper concentration by ultrapure water, then an excessive potassium permanganate solution is added for heating in boiling water, then a sodium oxalate solution is used for back dripping, the excessive sodium oxalate is added for back dripping by the potassium permanganate solution, and finally the determination is carried out according to the consumption of the potassium permanganate solution. The result is shown in fig. 2-4, fig. 2-4 can respectively measure the change of the colored component content and the total content of the algae-derived dissolved organic matter and the change of the organic pollution degree of the water body, after Bacillus tropicus ADOM11 bacteria are added, the ultraviolet-visible spectrum absorption curve of the algae-derived dissolved organic matter shows an obvious rapid degradation trend, and the total content (calculated by dissolved organic carbon) removal rate reaches 91.0% after 72h of culture; the chemical oxygen demand also shows a rapid reduction trend, and the organic pollution degree is removed to 82.9%.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.
SEQUENCE LISTING
<110> Shandong province fresh water fishery research institute (Shandong province fresh water fishery monitoring center)
<120> a tropical bacillus strain and its use
<130> 2021
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 1
<210> 2
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 2
tacggytacc ttgttacgac tt 22
<210> 3
<211> 1351
<212> DNA
<213> Bacillus tropicus
<400> 3
gttagcggcg gacgggtgag taacacgtgg gtaacctgcc cataagactg ggataactcc 60
gggaaaccgg ggctaatacc ggataacatt ttgaaccgca tggttcgaaa ttgaaaggcg 120
gcttcggctg tcacttatgg atggacccgc gtcgcattag ctagttggtg aggtaacggc 180
tcaccaaggc aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga 240
cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 300
gacggagcaa cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg 360
aagaacaagt gctagttgaa taagctggca ccttgacggt acctaaccag aaagccacgg 420
ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggaattattg 480
ggcgtaaagc gcgcgcaggt ggtttcttaa gtctgatgtg aaagcccacg gctcaaccgt 540
ggagggtcat tggaaactgg gagacttgag tgcagaagag gaaagtggaa ttccatgtgt 600
agcggtgaaa tgcgtagaga tatggaggaa caccagtggc gaaggcgact ttctggtctg 660
taactgacac tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 720
acgccgtaaa cgatgagtgc taagtgttag agggtttccg ccctttagtg ctgaagttaa 780
cgcattaagc actccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg 840
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 900
aggtcttgac atcctctgaa aaccctagag atagggcttc tccttcggga gcagagtgac 960
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020
gcgcaaccct tgatcttagt tgccatcatt aagttgggca ctctaaggtg actgccggtg 1080
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1140
acacgtgcta caatggacgg tacaaagagc tgcaagaccg cgaggtggag ctaatctcat 1200
aaaaccgttc tcagttcgga ttgtaggctg caactcgcct acatgaagct ggaatcgcta 1260
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320
cacaccacga gagtttgtaa cacccgaagt c 1351
Claims (9)
1. A tropical Bacillus strain is characterized in that the tropical Bacillus strain is tropical Bacillus ADOM11, which is preserved in China general microbiological culture Collection center at 25.1.25.2021, the preservation address is No. 3 Siro No. 1 of Beijing Korean district, the preservation number is CGMCC No.21730, and the strain is classified and named as tropical Bacillus tropicus.
2. Use of the strain of bacillus tropicalis according to claim 1 for remediating organic pollution of a water body of a water bloom.
3. Use according to claim 2, wherein bacillus tropicalis ADOM11 is applied to degrade algae-derived dissolved organic matter.
4. The use according to claim 3, wherein the bacillus tropicalis ADOM11 bacterial liquid is added to a water body containing algae-derived dissolved organic matter for degradation.
5. The use of claim 4, wherein the bacillus tropicalis ADOM11 bacterial liquid is obtained by inoculating bacillus tropicalis ADOM11 into a sterilized nutrient broth culture solution, shaking for culture, and centrifuging and suspending in physiological saline.
6. The use according to claim 4, wherein the viable count of the liquid of Bacillus tropicalis ADOM11 is 5 x 108-6×108one/mL.
7. The use of claim 4, wherein the bacillus tropicalis ADOM11 bacterial liquid is added into a water body containing algae-derived dissolved organic matters for degradation according to the volume ratio of 3-10%.
8. Use according to claim 4, wherein the degradation time is more than 12 h.
9. The use according to claim 4, wherein the viable count of the liquid of Bacillus tropicalis ADOM11 is 5.53 x 108-6×108Adding the strain per mL of the organic matter dissolved in the algae source into a water body containing the algae source dissolved organic matter according to the volume ratio of 5%, and degrading for more than 72 h.
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| CN113481128A (en) * | 2021-08-01 | 2021-10-08 | 重庆工商大学 | Bacillus tropicalis and application thereof in reduction of Cr (VI) |
| CN113617829A (en) * | 2021-08-01 | 2021-11-09 | 重庆工商大学 | Biological oil removal method for soil containing heavy metals and waste engine oil |
| CN113680812A (en) * | 2021-08-01 | 2021-11-23 | 重庆工商大学 | Bacillus tropicalis with Cr (VI) removal and petroleum hydrocarbon degradation and application thereof |
| CN114752519A (en) * | 2022-02-25 | 2022-07-15 | 中国水产科学研究院南海水产研究所 | Method for improving fermentation quality of marine fish by utilizing fermented tropical bacillus |
| CN114854637A (en) * | 2022-05-24 | 2022-08-05 | 华南农业大学 | Phosphorus-dissolving growth-promoting bacillus tropicalis SG15, biological agent and application of biological agent in potato seed dressing agent |
| CN117535210A (en) * | 2024-01-09 | 2024-02-09 | 青岛农业大学 | Salt-tolerant growth-promoting bacterium, microbial inoculum, preparation method of microbial inoculum and application of microbial inoculum |
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| CN113481128A (en) * | 2021-08-01 | 2021-10-08 | 重庆工商大学 | Bacillus tropicalis and application thereof in reduction of Cr (VI) |
| CN113617829A (en) * | 2021-08-01 | 2021-11-09 | 重庆工商大学 | Biological oil removal method for soil containing heavy metals and waste engine oil |
| CN113680812A (en) * | 2021-08-01 | 2021-11-23 | 重庆工商大学 | Bacillus tropicalis with Cr (VI) removal and petroleum hydrocarbon degradation and application thereof |
| CN113481128B (en) * | 2021-08-01 | 2023-06-13 | 重庆工商大学 | Bacillus tropicalis and application thereof in reduction of Cr (VI) |
| CN114752519A (en) * | 2022-02-25 | 2022-07-15 | 中国水产科学研究院南海水产研究所 | Method for improving fermentation quality of marine fish by utilizing fermented tropical bacillus |
| CN114752519B (en) * | 2022-02-25 | 2022-09-27 | 中国水产科学研究院南海水产研究所 | Method for improving fermentation quality of marine fish by utilizing fermented tropical bacillus |
| CN114854637A (en) * | 2022-05-24 | 2022-08-05 | 华南农业大学 | Phosphorus-dissolving growth-promoting bacillus tropicalis SG15, biological agent and application of biological agent in potato seed dressing agent |
| CN114854637B (en) * | 2022-05-24 | 2023-09-05 | 华南农业大学 | Phosphorus-dissolving growth-promoting bacillus tropicalis SG15 and biological microbial agent and application thereof in potato seed dressing agent |
| CN117535210A (en) * | 2024-01-09 | 2024-02-09 | 青岛农业大学 | Salt-tolerant growth-promoting bacterium, microbial inoculum, preparation method of microbial inoculum and application of microbial inoculum |
| CN117535210B (en) * | 2024-01-09 | 2024-04-12 | 青岛农业大学 | Salt-tolerant growth-promoting bacterium, microbial inoculum, preparation method of microbial inoculum and application of microbial inoculum |
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