CN110438040B - Bacillus licheniformis strain and application thereof - Google Patents
Bacillus licheniformis strain and application thereof Download PDFInfo
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- CN110438040B CN110438040B CN201910659009.6A CN201910659009A CN110438040B CN 110438040 B CN110438040 B CN 110438040B CN 201910659009 A CN201910659009 A CN 201910659009A CN 110438040 B CN110438040 B CN 110438040B
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- C—CHEMISTRY; METALLURGY
- 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
- C02F3/02—Aerobic processes
- C02F3/12—Activated sludge processes
- C02F3/1236—Particular type of activated sludge installations
- C02F3/1263—Sequencing batch reactors [SBR]
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- C—CHEMISTRY; METALLURGY
- 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
- C02F3/30—Aerobic and anaerobic processes
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- C—CHEMISTRY; METALLURGY
- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
Bacillus licheniformis strain FA6 (Bacillus licheniformis strain) ((Bacillus licheniformis strain))Bacillus lincheniformis) And is preserved in China center for type culture Collection in 2018, 6 and 29 months with the preservation number of CCTCC NO: m2018410. The bacillus licheniformis FA6 can be respectively inoculated into a seed culture medium and a sterilized sewage fermentation culture medium to obtain a fermentation inoculum thereof, and the fermentation inoculum can be further applied to a sewage treatment process. The bacillus licheniformis FA6 and the fermentation inoculant thereof can prevent and regulate sludge bulking by a quorum sensing method, have the advantages of low cost, small pollution and the like, and are worthy of wide popularization and application.
Description
Technical Field
The invention relates to a microbial preparation and application thereof in the field of sewage treatment, and more particularly relates to a bacillus licheniformis strain FA6 and application thereof.
Background
Along with the rapid improvement of the urbanization degree of China, the generation of a large amount of domestic sewage and industrial wastewater generates great pressure on sewage treatment plants. The activated sludge process is the most widely applied treatment method in the current sewage treatment, and has the characteristics of low operation cost, large water treatment amount and the like, but the activated sludge process is easy to generate sludge bulking phenomenon.
The sludge bulking phenomenon refers to the phenomena of loose sludge floc structure, difficult sludge-water separation and the like, and is a common and difficult-to-treat problem in sewage treatment plants at home and abroad at present. The sludge bulking is mainly caused by excessive propagation of filamentous bacteria, and the flocculation property and the settleability of the sludge are greatly reduced due to the propagation of a large amount of filamentous bacteria, so that the whole sewage treatment process and the effect are influenced.
At present, no effective preventive measures for the process flow are available to solve the problem of sludge bulking, and emergency measures can influence the whole sewage treatment flow and the downstream water treatment and damage the operation period of the system.
At present, the existing sludge bulking treatment measures are basically carried out by using chemical agents, and are usually treated by using bactericides and flocculating agents, wherein the bactericides comprise chlorine preparations such as ozone, hydrogen peroxide, chlorine and the like; the flocculating agent comprises aluminum sulfate, ferric chloride and the like, the chemical agent has certain economic consumption and higher cost, and the chemical agent also generates certain pollution. Ecological regulation is a green and environment-friendly method advocated in recent years, which can save a large amount of economic energy consumption and greatly reduce environmental pollution. The ecological regulation method is to perform population inhibition of filamentous fungi by quorum sensing. Quorum Sensing (QS) refers to the regulation of expression of specific genes in microorganisms by signal molecules.
Disclosure of Invention
In order to solve the defect of sludge bulking caused by the existing chemical agent treatment, the invention aims to provide the bacillus licheniformis FA6 capable of inhibiting excessive propagation of filamentous bacteria to cause sludge bulking through quorum sensing.
The Bacillus licheniformis strain FA6 (Bacillus licheniformis FA 6) is preserved in the China center for type culture Collection in 6 months and 29 months in 2018, and the preservation number is CCTCC NO: m2018410, the preservation address is Wuhan university in China.
The gene sequence of 16S rRNA of the bacillus licheniformis FA6 is shown in SEQ ID No. 1.
The invention also provides a fermentation inoculum of the bacillus licheniformis FA 6.
The preparation method of the bacillus licheniformis FA6 fermentation inoculant comprises the steps of inoculating bacillus licheniformis FA6 to a seed culture medium, culturing at the culture temperature of 37 ℃ and the rotation speed of 130rpm for 24 hours, then inoculating the bacillus licheniformis FA6 to a sterilized sewage fermentation culture medium for amplification culture, and culturing at the culture temperature of 37 ℃ for 24 hours to obtain the bacillus licheniformis FA6 fermentation inoculant; the seed culture medium contains 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1000 mL of distilled water; the sterilized sewage fermentation medium contains 1.0 percent of peptone, 0.5 percent of yeast powder, 1.0 percent of NaCl, 0.2 percent of 25 percent concentration manganese sulfate, 0.3 percent of monopotassium phosphate, 0.3 percent of calcium carbonate, 0.1 percent of ammonium sulfate, 0.05 percent of potassium chloride, 1 percent of calcium phosphate, 0.05 percent of magnesium sulfate, 800 mL of distilled water and 200 mL of sterilized pool water of a pretreated sewage pool, wherein the percentages are weight to volume ratio.
The invention also provides application of the bacillus licheniformis FA6 fermentation inoculant in sewage treatment.
The invention has the advantages of
The bacillus licheniformis FA6 can achieve the purpose of inhibiting the overgrowth of bacteria by generating lipopeptide substances and interfering a quorum sensing system of the bacteria, is an environment-friendly ecological regulation and control method, and has the characteristics of low economic cost, small environmental pollution and the like. In addition, the bacillus licheniformis FA6 serving as a facultative anaerobe has the characteristics of high temperature resistance, acid and alkali resistance and the like, is strong in stress resistance, and has the capability of producing spores, so that the bacillus licheniformis FA6 can be well applied to various sewage treatment equipment.
Detailed Description
The present invention will be described in further detail with reference to examples, and reagents and consumables used in the present invention are commercially available conventional products unless otherwise specified.
Example 1 isolation screening and characterization of Bacillus licheniformis FA6
(1) Separation and screening of bacillus licheniformis FA 6:
screening suspected strains with quorum sensing signal molecule inhibition effect by using a filter paper method, collecting a plurality of strains of bacillus from soil, sewage and animal intestinal tracts respectively, inoculating the collected bacillus into liquid LB culture medium to be cultured for 24 hours at the culture temperature of 37 ℃ and the rotation speed of 130rpm, filtering by using a 0.22 mu m bacterial filter membrane to obtain 100 mL of supernatant, adding the supernatant into a separating funnel, extracting by using 500 mL of ethyl acetate to repeatedly oscillate, collecting an emulsion layer, dripping 10 mu L of the supernatant into 5 mm filter paper and airing in a sterile operating platform, and repeating the adding and airing for three times. And coating the activated staphylococcus aureus on a solid LB culture medium, pressing a prepared filter paper sheet on the culture medium, placing the culture medium in a constant-temperature incubator, culturing for 24 hours at 37 ℃, dripping ethyl acetate without crude extract on the filter paper sheet as a control group, and observing and recording the diameter of a bacteriostatic circle in the culture medium. Through the method, a bacillus licheniformis strain capable of generating a bacteriostatic circle is screened, is named as FA6, is preserved in China center for type culture Collection, and has the preservation number of CCTCC NO: m2018410, wherein the preservation time is 2018, 6 and 29 months, and the preservation place is Wuhan university in China.
(2) Identification of bacillus licheniformis:
carrying out PCR identification on the screened strains by using the universal primers of the bacteria: template DNA was extracted according to the protocol of a bacterial DNA extraction kit (from DN1101, Edley Biotech, Beijing). Upstream primer 27F using 16S rRNA conserved sequence: 5 '-AGAGTTTGATCMTGGCTCAG-3' (shown as SEQ ID No. 2) and a downstream primer 1492R: 5 '-TACGGYTACCTTGTTACGACTT-3' (shown in SEQ ID No. 3), PCR amplification is performed on the 16S rRNA gene fragment of Bacillus licheniformis FA6, an amplification product is recovered by using an agarose gel purification kit (purchased from DR0101, Erdela bioscience, Beijing), and the recovered product is ligated with pMD-18T vector and transformed into DH5 alpha competent cells, spread on 57 mg/mL ampicillin LB medium, and cultured at 37 ℃ for 10 h. Transformants were randomly selected and purified using vector universal primers M13R: 5 '-CAGGAAACAGCTATGACC-3' (shown as SEQ ID No. 4) and M13F: 5 '-TGTAAAACGACGGCCAGT-3' (shown as SEQ ID No. 5), detecting and sequencing to obtain a 1444bp fragment, wherein the accession number of the fragment in GenBank is MG428771.1, and sequencing to obtain the strain belonging to Bacillus licheniformis, wherein the 16S rRNA gene sequence of the strain is shown as SEQ ID No. 1.
The ampicillin LB medium comprises 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 57 mg/mL ampicillin.
Example 2 determination of quorum sensing by Bacillus licheniformis FA6
The method for determining quorum sensing of bacillus licheniformis FA6 obtained in example 1 specifically comprises the following steps:
(1) extraction of lipopeptides
Inoculating bacillus licheniformis FA6 stored in a refrigerator at the temperature of-80 ℃ into a liquid LB culture medium, culturing at the temperature of 37 ℃ and the rotation speed of 130rpm for 12 h for activation, inoculating an activated FA6 strain into 100 mL of LB liquid culture medium, culturing at 37 ℃ and 180 rpm in a constant temperature incubator for 24 h, inoculating 1 mL of bacterial liquid into 100 mL of Landy culture medium, culturing at 37 ℃ and 180 rpm in the constant temperature incubator for 48 h, centrifuging at 12000 rpm for 30 min, obtaining supernatant, regulating the pH to 2.0 by using HCl, precipitating at 4 ℃ overnight, centrifuging at 12000 rpm for 30 min again, retaining the precipitate, adding an appropriate amount of methanol to dissolve the precipitate, centrifuging at 12000 rpm for 10 min, obtaining supernatant, and regulating the concentration to 1 mg/mL by using an appropriate amount of methanol to obtain the lipopeptide crude extract.
(2) Separation and purification of lipopeptide substances
The lipopeptide extract of the FA6 strain was filtered through a 0.22 μm bacterial filter and analyzed and compared by fast protein liquid chromatography. The mobile phase solution A contains 2% of acetonitrile and 0.065% of trifluoroacetic acid, the mobile phase solution B contains 80% of acetonitrile and 0.05% of trifluoroacetic acid, the gradient elution process is that the mobile phase A is 100% to 0 within 49.86 min, the detection wavelength is 215 nm, and the flow rate is 1 mL/min.
(3) Bacteriostatic assay for lipopeptides
And (3) determining the bacteriostatic activity of the FA6 strain lipopeptide extract by taking staphylococcus aureus as a target bacterium. The lipopeptide extract was filtered through a 0.22 μm bacterial filter. And adding 50 mu L of lipopeptide extract onto a filter paper sheet, drying, placing on an LB culture medium inoculated with staphylococcus aureus by a coating inoculation method, and repeating the experiment for three times. The control group used a filter paper sheet made of physiological saline.
Liquid LB medium: 10 g/L of tryptone, 5 g/L of yeast extract and 10 g/L of NaCl.
Solid LB medium: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl and 15 g/L of agar.
Landy medium: glucose 20 g/L, L-glutamic acid 5 g/L, MgSO4·7H2O 1.02 g/L，KH2PO4 1.0 g/L，KCL 0.5 g/L，FeSO4·7H2O 0.15mg/L，MnSO4·H2O 5.0 mg/L。CuSO4·5H2O 0.16 mg/L。
Results of the experiment
No zone of inhibition was found around the filter paper of the control LB plate.
The growth of staphylococcus aureus is found to be inhibited around the filter paper sheets of the LB flat plate of the experimental group, and the diameter of the average inhibition zone is 12 mm.
Therefore, bacillus licheniformis can achieve the purpose of inhibiting the overgrowth of bacteria by producing lipopeptide substances and interfering the quorum sensing system of bacteria.
EXAMPLE 3 preparation of Bacillus licheniformis FA6 fermentation inoculum
(1) Seed culture medium
10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1000 mL of distilled water.
(2) Sterilized sewage fermentation culture medium
Peptone 1.0%, yeast powder 0.5%, NaCl 1.0%, 25% manganese sulfate 0.2%, monopotassium phosphate 0.3%, calcium carbonate 0.3%, ammonium sulfate 0.1%, potassium chloride 0.05%, calcium phosphate 1%, magnesium sulfate 0.05%, distilled water 800 mL, and pretreatment sewage pool sterilizing pool water 200 mL, wherein the percentages are weight to volume ratio.
Inoculating the bacillus licheniformis FA6 to a seed culture medium, culturing at the culture temperature of 37 ℃ and the rotation speed of 130rpm for 24 h, inoculating to a sterilized sewage fermentation culture medium, performing amplification culture, and culturing at the culture temperature of 37 ℃ for 24 h to obtain the bacillus licheniformis FA6 fermentation inoculum.
Example 4 application of Bacillus licheniformis FA6 fermentation inoculant in SBR water treatment equipment
(1) Experimental device
A laboratory SBR reactor is adopted, the effective volume of the laboratory SBR reactor is 20L, the operation period of the laboratory SBR reactor comprises five parts of water inlet, stirring, aeration, precipitation and water drainage, the operation time is respectively 2 h, 1.5 h, 0.5 h and 0.5 h, and the operation period is 6 h in total. The experimental temperature was 20. + -. 5 ℃.
(2) Construction of filamentous bacterium sludge bulking
The experimental sewage comes from sewage deteriorated in settlement of a sewage treatment plant in certain city in Wuhan. The experimental group was supplemented with 1% by volume of bacillus licheniformis FA6 fermentation broth and the control group with 1% by volume of sterile fermentation medium. Detecting a Sludge Volume Index (SVI) of the sludge and the sludge particle size; observing the abundance of the filamentous fungi by a microscope.
Results of the experiment
Sludge Volume Index (SVI)
The control group developed sludge bulking at the 30 th cycle and the Sludge Volume Index (SVI) increased to 183 mL/g and reached 213 mL/g at the maximum at the 70 th cycle, while the experimental group was 128 mL/g at the 30 th cycle and 124 mL/g at the 70 th cycle.
Sludge grain size
The samples were examined for sludge particle size using a laser particle sizer (Beckman) with the control group mean particle size at 30 cycles being 96 μm and decreasing to 59 μm at 70 run cycles, while the experimental group mean particle size at 30 cycles being 132 μm and 119 μm at 70 run cycles.
Abundance of filamentous fungi
The abundance of the filamentous fungi in the control group is 4 grades at the 30 th cycle, the enrichment is reduced to 5 grades at the 70 th running cycle, and a large amount of filamentous fungi hyphae protrude from sludge flocs through microscopic examination. Whereas the abundance of the experimental group of filamentous fungi was grade 2 at cycle 30 and increased to grade 4 at cycle 70.
Abundance level of filamentous fungi
Stage 0: no filamentous fungi were found on all flocs.
Stage 1: very rarely, filamentous fungi are found on individual flocs.
Stage 2: some, not all floes have filamentous bacteria on them.
Stage 3: generally, all flocs have hyphae on them, but at a lower density. Each floc had 1 to 5 hyphae.
And 4, stage: more, all the flocs have hyphae and medium density. There were 5 to 20 hyphae on each floc.
Stage 5: rich in mycelium and high in density. Hyphae were more than 20 per floc.
Stage 6: a large number of hyphae form a silk net.
Example 5 application of Bacillus licheniformis FA6 fermentation inoculum in AO experiment pool
(1) Experimental device
An outdoor self-built AO reactor is adopted for carrying out experiments and comprises an anoxic zone, an aerobic zone, a sludge multi-point backflow device, a multi-point water inlet device and the like. Sampling and detecting once a day. The experimental temperature was 20 ℃.
(2) Construction of filamentous bacterium sludge bulking
The experimental sewage comes from sewage deteriorated in settlement of a sewage treatment plant in certain city in Wuhan. The experimental group was supplemented with 1% by volume of bacillus licheniformis FA6 fermentation broth and the control group with 1% by volume of sterile fermentation medium. Detecting a Sludge Volume Index (SVI) of the sludge and the sludge particle size; observing the abundance of the filamentous fungi by a microscope.
Results of the experiment
Sludge Volume Index (SVI)
The control group developed sludge bulking at day 20, and the Sludge Volume Index (SVI) increased to 163 mL/g, up to 201 mL/g at day 35, while the experimental group did not increase to 150 mL/g throughout the run.
Sludge grain size
The mean particle size of the control group was 96 μm on day 20 and decreased to 59 μm on day 35, while the mean particle size of the experimental group was 132 μm on day 20 and 119 μm on day 35.
Abundance of filamentous fungi
The abundance of the filamentous fungi in the control group is 5 grades at day 20, and is reduced to 6 grades at day 35, and a large amount of filamentous fungi hyphae protrude from sludge flocs through microscopic examination. Whereas the abundance of the experimental group of filamentous fungi was grade 3 on both day 20 and day 35.
In conclusion, the bacillus licheniformis FA6 provided by the invention can be used as an ecological additive in the water treatment process of an activated sludge process to prevent and regulate the sludge bulking problem caused by the overgrowth of filamentous bacteria.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Sequence listing
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<120> Bacillus licheniformis strain and application thereof
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gggaaaccgg ggctaatacc ggatgcttga ttgaaccgca tggttcaatt ataaaaggtg 180
gcttttagct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240
ctcaccaagg caacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360
tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaaactc tgttgttagg 420
gaagaacaag taccgttcga atagggcggt accttgacgg tacctaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540
gggcgtaaag cgcgcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600
gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660
tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720
gtaactgacg ctgaggcgcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780
cacgccgtaa acgatgagtg ctaagtgtta gagggtttcc gccctttagt gctgcagcaa 840
acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960
caggtcttga catcctctga caaccctaga gatagggctt ccccttcggg ggcagagtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt 1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200
cacacgtgct acaatgggca gaacaaaggg cagcgaagcc gcgaggctaa gccaatccca 1260
caaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct 1320
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Claims (3)
1. The application of a Bacillus licheniformis fermentation inoculant in sewage treatment is characterized in that the Bacillus licheniformis fermentation inoculant is a fermentation inoculant containing a Bacillus licheniformis strain FA6, the Bacillus licheniformis strain FA6 is preserved in the China Center for Type Culture Collection (CCTCC) in 2018, 6 and 29 months, and the preservation number is CCTCC NO: m2018410.
2. The use of the Bacillus licheniformis zymogen agent of claim 1 in SBR batch activated sludge process water treatment equipment.
3. The use of the Bacillus licheniformis zymogen of claim 1 in AO anaerobic aerobic process laboratory tanks.
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| CN107603939A (en) * | 2017-11-03 | 2018-01-19 | 钟华 | A kind of salt tolerant bacterium culture medium nutritional agents and preparation method thereof and acclimation method and application |
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