CN114574409B - Pseudobacillus, fermentation product thereof and application thereof in algae solubilization - Google Patents
Pseudobacillus, fermentation product thereof and application thereof in algae solubilization Download PDFInfo
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- CN114574409B CN114574409B CN202210489455.9A CN202210489455A CN114574409B CN 114574409 B CN114574409 B CN 114574409B CN 202210489455 A CN202210489455 A CN 202210489455A CN 114574409 B CN114574409 B CN 114574409B
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Abstract
The invention discloses a pseudobacillus, a fermentation product thereof and application thereof in algae lysis, belonging to the technical field of microorganisms. The preservation number of the pseudobacillus 630 is as follows: GDMCC No: 62328, Strain 630 against Cephalosporium Tomentosum (Cystospodophyllum nodosum) ((R))Raphidocelis subcapitata) And, Yucca (F.) KuntzeUlothrixsp., Chlorella pyrenoidosa (Chlorella pyrenoidosa, and/or Chlorella pyrenoidosa)Chlorella pyrenoidosa) And Tetrastigmata slantwise: (Tetradesmus obliquus) Has certain algae dissolving effect, and shows that the algae dissolving spectrum is rich, and the algae dissolving agent has strong application potential in the aspect of biological agents for controlling algal bloom.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a pseudobacillus, a fermentation product thereof and application thereof in algae lysis.
Background
Water resources are the material basis on which humans live. The eutrophication of the water body causes the abnormal mass propagation of part of algae and other aquatic organisms to form cyanobacterial bloom, which can cause the death of fishes and shrimps, further cause the water body pollution and the blockage of water channels, and have serious influence on the production and the life of human beings. In addition, more than 80% of cyanobacterial bloom can detect a secondary metabolite, namely microcystin. The microcystin has high structural stability, is heat-resistant, is not easy to decompose by boiling water, and is a strong carcinogen besides generating toxicity directly to fishes, people and livestock, especially an important cause of liver cancer.
An algicidal bacterium (algicidal bacterium) is a bacterium which lyses in a direct or indirect manner to destroy the cell structure of algae or inhibit the growth of algaeLong bacteria. In recent years, researchers separate and screen algicidal bacteria, apply the algicidal bacteria to the algae bloom prevention and control of eutrophic fishponds, shrimp culture ponds, lakes, riverways and other environments, effectively reduce the number of algae cells in water, and have wide application prospects. The most commonly reported phycolytica include Bacillus (B.sub.), (B.sub.)Bacillusspp.), Streptomyces (Streptomyces)Streptomycesspp.) and Pseudomonas (Pseudomonasspp.) and the like, and Pseudomonas (B.pseudomonad) ((B.pseudomonad)Fictibacillussp.) was reported less in algicidal action.
Disclosure of Invention
Therefore, the invention aims to provide a strain of pseudobacillus (A), (B), (C)Fictibacillussp.) 630 and its use in lysing algae.
The first purpose of the invention is to provide a strain of pseudobacillus (A), (B), (C)Fictibacillussp.) 630, accession number: GDMCC No: 62328.
it is a second object of the present invention to provide a preparation comprising the above-mentioned Pseudomonas 630 or a fermentation culture thereof.
Preferably, the fermentation culture is a fermentation broth obtained by culturing the pseudobacillus 630 or a fermentation broth from which the bacteria are removed, such as by centrifuging and filtering the fermentation broth to obtain a sterile filtrate.
Preferably, the culturing comprises the following steps: the strain of the pseudomonas 630 is subjected to amplification culture by using an R2A culture medium, and then the bacterial liquid is transferred to an R2A liquid culture medium and cultured in a shaking table with the rotating speed of 180R/min at 30 ℃.
More preferably, the bacterial liquid is transferred to the liquid culture medium at a volume ratio of 1: 50.
More preferably, the composition of the R2A medium is as follows: 0.25 g/L tryptone, 0.5 g/L yeast extract, 0.5 g/L acid hydrolyzed casein, 0.25 g/L peptone, 0.5 g/L glucose, 0.5 g/L soluble starch, 0.3 g/L sodium pyruvate, 0.1 g/L magnesium sulfate, 0.3 g/L dipotassium hydrogen phosphate, and pH 7.2.
The third purpose of the invention is to provide the application of the pseudobacillus 630 or the preparation in inhibiting the growth of algae.
Preferably, the algaeThe class is cladocera (A) and (B)Raphidocelis subcapitata) And, Yucca (Ulothrixsp., Chlorella pyrenoidosa (Chlorella pyrenoidosa, and/or Chlorella pyrenoidosa)Chlorella pyrenoidosa) Or Tetrastigmata slantwise: (Tetradesmus obliquus）。
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, a strain of the pseudomonas 630 with algae-lysing diversity is screened out, the strain 630 can inhibit the growth of cephalosporium juveniles, filaria, chlorella pyrenoidosa and tetragonia, the removal rate of chlorophyll a is 71.72%, 29.85%, 29.23% and 13.18%, and the algae-lysing type is not reported before, so that the algae-lysing spectrum of the pseudomonas is possibly richer, and the pseudomonas has strong application potential in biological agents.
Deposit description
According to the inventionFictibacillussp, 630, deposited at 14.4.2022 in Guangdong province microbial culture Collection (GDMCC), and addressed to No. 59, No. 5, of No. 100, Ministry of Toeli, Guangzhou, and the postal code is: 510070, accession number: GDMCC No: 62328.
drawings
FIG. 1 shows Pseudobacillus (Fictibacillussp.) 630.
FIG. 2 shows Pseudobacillus sp. (Fictibacillussp.) 630.
FIG. 3 shows Pseudobacillus sp. (Fictibacillussp.) 630 algae lysing effect on different algae species.
Detailed Description
The following is a further description of the present invention with reference to examples, but not intended to limit the present invention.
Example 1 Pseudobacillus sp. (Fictibacillussp.) 630 isolation, identification and cultivation
Pseudobacillus of the present invention: (A), (B)Fictibacillussp.) 630 is isolated and purified from Poyang lake of south China agriculture university in Tianhewa area, Guangzhou, Guangdong province, and comprises the following steps:
(1) separation: collect Poyang of south China agricultural universityLake water sample, diluting to 10% 5 、10 6 、10 7 Then spread on the algae powder inorganic salt culture medium (1 g/L K) 2 HPO 4 ，0.5 g/L MgSO 4 ，0.5 g/L NH 4 Cl，0.5 g/L KNO 3 0.1% of trace elements, 0.1% of vitamins and 1% of high-purity spirulina powder (purchased from Wanbang industry Co., Ltd. of Henan), wherein the formulation of the trace elements is 10 mL/L HCl and 1.5 g/L FeCl 2 ·4H 2 O，190 mg/L CoCl 2 ·6H 2 O，100 mg/L MnCl 2 ·4H 2 O，mg/L，70 mg/L ZnCl 2 ，62 mg/L H 2 BO 4 ，36 mg/L Na 2 MoO 4 ·2H 2 O，24 mg/L NiCl 2 ·6H 2 O，17 mg/L CuCl 2 ·2H 2 O, the formula of the vitamins is 2mg/L folic acid, 10 mg/L vitamin B6, 5 mg/L vitamin B2, 2mg/L vitamin H, 5 mg/L vitamin B1, 5 mg/L vitamin B5, 0.1 mg/L vitamin B12, 5 mg/L nicotinic acid, 5 mg/L p-aminobenzoic acid and 5 mg/L lipoic acid, the solvent is water, the components are added into water, and the mixture is uniformly mixed and sterilized to obtain the strain to be tested 630) through separation.
(2) And (3) identification: the colonies were amplified from the 16S rRNA gene fragment of a single bacterium using bacterial universal primers 1492R and 27F, and sequenced as shown in FIG. 1 (Biotechnology, Shanghai, Ltd.). The two obtained positive and negative sequences are spliced by using Contig Express software and then are compared and analyzed in a GenBank database. Pseudobacillus bacteria (A), (B), (C)Fictibacillussp.) 630 molecular classification: the 16S rRNA gene sequence of the strain 630 is shown as SEQ ID NO.1, and the strains 630 and 630Fictibacillus obuense47001 with similarity of 98.81%, preliminarily identifying that the strain 630 belongs to Pseudomonas, and naming it as PseudomonasFictibacillussp, 630, deposited at 14.4.2022 in Guangdong province microbial culture Collection (GDMCC), and addressed to No. 59, No. 5, of Mirabilite 100, Guangzhou, zip code: 510070, accession number: GDMCC No: 62328.
(3) culturing: the strain is cultured in R2A medium (0.25 g/L tryptone, 0.5 g/L yeast extract powder, 0.5 g/L acid hydrolyzed casein, 0.25 g/L peptone, 0.5 g/L glucose)Performing amplification culture on soluble starch of 0.3 g/L, sodium pyruvate of 0.1 g/L, magnesium sulfate of 0.3 g/L and dipotassium hydrogen phosphate of 0.3 g/L at the pH value of 7.2), transferring 100 mu L of bacterial liquid into a test tube filled with 5 mL of R2A liquid culture medium, culturing at 30 ℃ in a shaking table at the rotating speed of 180R/min, and measuring the light absorption value at the wavelength of 600 nm (per hour) ((the concentration of the bacterial liquid is measured)OD 600 ) Let 3 be parallel. To be provided withOD 600 The growth curve is plotted with the ordinate and time on the abscissa. The growth curve of strain 630 is shown in FIG. 2.
Example 2 Pseudobacillus bacteria: (Fictibacillussp.) 630 broad algae lysing spectrum
The slant-grown Tetrastigmata Alternata (B) ((B))Tetradesmus obliquus) And Chlorella pyrenoidosa: (A)Chlorella pyrenoidosa) Cytospora brachypodium (Leptospira Tomentosa, Leptospira interrogans, and Haptospira interrogansRaphidocelis subcapitata) And the Dioscorea (A), (B), (C)Ulothrixsp.) were inoculated into BG11 medium for 10 days, and the test strains of Pseudomonas 630 (see the culture method (3) in example 1, using the bacterial solution of Pseudomonas 630 in stationary phase) were inoculated into BG11 medium containing Tetradesmus clinopodii, Chlorella pyrenoidosa, Cyanotheca racemosa, and Cyanopsis (the ratio of the algal solution to the volume is 1: 9) the cells were cultured at 25 ℃ for 4 days at 12 h day/12 h night and 2500 lx, and 3 cells were placed in each group in parallel. The Chl-a concentration of the algae culture solution is determined according to an acetone extraction method in spectrophotometry for measuring chlorophyll in water (formula (1)), and the algae-lysing rate R (Chl-a removal rate) is calculated according to a formula (2).
Formula (1)
Wherein Chl-a is the chlorophyll a concentration (mg/L) of the sample;ODabsorbance at different wavelengths; l is the optical distance (cm) of the cuvette; v 1 Volume of acetone extract (mL); v 2 Is the sample volume (L).
Wherein, R is chlorophyll a removal rate (%); chl-a Control group The chlorophyll a concentration (mg/L) of the algae liquid after the algae liquid and the sterile R2A culture medium are co-cultured; chl-a Experimental group The chlorophyll a concentration (mg/L) of the algae liquid after co-culture with the bacterial liquid.
Pseudobacillus bacteria (A), (B), (C)Fictibacillussp.) 630 inhibits cladocephalusRaphidocelis subcapitata) And, Yucca (Ulothrixsp.), Chlorella pyrenoidosa (Chlorella pyrenoidosa) Or Tetrastigmata slantwise: (Tetradesmus obliquus) The chlorophyll-a removal rates were 71.72%, 29.85%, 29.23% and 13.18%, respectively (fig. 3).
The above embodiment examples illustrate the different implementation procedures of the present invention in detail, but the embodiments of the present invention are not limited thereto. The purpose of the present invention can be achieved by those skilled in the art according to the disclosure of the present invention.
It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the principles of the invention, and it is intended that such changes and modifications also fall within the scope of the appended claims.
Sequence listing
<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)
<120> pseudobacillus, fermentation product thereof and application thereof in algae lysis
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1389
<212> DNA
<213> Pseudomonas 630(Fictibacillus sp.)
<400> 1
cctctgattt agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagacgggga 60
taacttcggg aaaccgaagc taataccgga taataaagag aaactcctgt ttcttttttg 120
aaagtcggtt tcggctgacg cttacagatg ggcccgcggc gcattagcta gttggtgagg 180
taacggctca ccaaggccac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg 240
actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcg gcaatggacg 300
aaagtctgac cgagcaacgc cgcgtgagcg atgaaggcct tcgggtcgta aagctctgtt 360
gtcagagaag aacaagtacc ggagtaactg ccggtacctt gacggtacct gaccagaaag 420
ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggaa 480
ttattgggcg taaagcgcgc gcaggcggtt ccttaagtct gatgtgaaag cccacggctc 540
aaccgtggag ggtcattgga aactggggaa cttgagtgca ggagagaaaa gtggaattcc 600
acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcggcttttt 660
ggcctgtaac tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg 720
tagtccacgc cgtaaacgat gagtgctagg tgttgggggg ttccaccctc agtgctgacg 780
ttaacacatt aagcactccg cctggggagt acggccgcaa ggctgaaact caaaggaatt 840
gacgggggcc cgcacaagca gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 900
taccaggtct tgacatcctc tgaccactct agagatagag ctttcccctt cgggggacag 960
agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020
caacgagcgc aacccttgac cttagttgcc agcattcagt tgggcactct aaggtgactg 1080
ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1140
ggctacacac gtgctacaat gggtggtaca aagggttgca aagccgcgag gccgagccaa 1200
tcccaaaaag ccactctcag ttcggattgt aggctgcaac tcgcctacat gaagccggaa 1260
ttgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1320
gcccgtcaca ccacgagagt ttgtaacacc cgaagccggt ggggtaacct tttggagcca 1380
gccgtcgaa 1389
Claims (4)
1. Pseudobacillus (A), (B) and (C)Fictibacillussp.) 630, accession number: GDMCC No: 62328.
2. a preparation comprising the Pseudomonas 630 according to claim 1 or a fermentation culture thereof, wherein the fermentation culture is a fermentation broth containing the Pseudomonas 630 obtained by culturing the Pseudomonas 630.
3. The formulation of claim 2, wherein said culturing comprises the steps of: the strain is subjected to amplification culture by using an R2A culture medium, a bacterial liquid is transferred into an R2A liquid culture medium, and the strain is cultured in a shaking table with the rotating speed of 180R/min at 30 ℃;
the components of the R2A culture medium are as follows: 0.25 g/L tryptone, 0.5 g/L yeast extract, 0.5 g/L acid hydrolyzed casein, 0.25 g/L peptone, 0.5 g/L glucose, 0.5 g/L soluble starch, 0.3 g/L sodium pyruvate, 0.1 g/L magnesium sulfate, 0.3 g/L dipotassium hydrogen phosphate, and pH 7.2.
4. Use of the pseudobacillus 630 of claim 1 or the formulation of any one of claims 2-3 to inhibit the growth of algae, said algae being cephalosporium Tomentosum (Nostoc Tomentosa) ((R))Raphidocelis subcapitata) And, Yucca (Ulothrixsp.), Chlorella pyrenoidosa (Chlorella pyrenoidosa) Or Tetrastigmata slantwise: (Tetradesmus obliquus）。
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Citations (3)
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| CN109486705A (en) * | 2018-11-20 | 2019-03-19 | 河南农业大学 | The pale false bacillus strain X21 of one kind and its application |
| WO2019194062A1 (en) * | 2018-04-05 | 2019-10-10 | 合同酒精株式会社 | Enzyme derived from p aenibacillus pabuli and capable of producing galactooligosaccharide and method for producing galactooligosaccharide |
| KR20210119754A (en) * | 2020-03-25 | 2021-10-06 | 한국생명공학연구원 | Composition for controlling red tide comprising Fictibacillus sp. or their medium as effective component |
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| JP7546305B2 (en) * | 2020-01-14 | 2024-09-06 | 天津大学 | Xanthine amidohydrolase and uses thereof |
| CN111763643A (en) * | 2020-07-09 | 2020-10-13 | 中国科学院南京土壤研究所 | Compound flora for preventing and treating peanut root rot and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019194062A1 (en) * | 2018-04-05 | 2019-10-10 | 合同酒精株式会社 | Enzyme derived from p aenibacillus pabuli and capable of producing galactooligosaccharide and method for producing galactooligosaccharide |
| CN109486705A (en) * | 2018-11-20 | 2019-03-19 | 河南农业大学 | The pale false bacillus strain X21 of one kind and its application |
| KR20210119754A (en) * | 2020-03-25 | 2021-10-06 | 한국생명공학연구원 | Composition for controlling red tide comprising Fictibacillus sp. or their medium as effective component |
Non-Patent Citations (2)
| Title |
|---|
| Fictibacillus aquaticus sp. nov., isolated from downstream river water;Deepika Pal 等;《Int J Syst Evol Microbiol》;20180101;第68卷(第1期);第160-164页 * |
| 细菌对引发赤潮相关藻类的杀藻作用研究综述;高前程 等;《渔业研究》;20211231;第43卷(第4期);第426-435页 * |
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