CN112940971A - Delftia sp for regulating quorum sensing quenching and separation method and application thereof - Google Patents
Delftia sp for regulating quorum sensing quenching and separation method and application thereof Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- C—CHEMISTRY; METALLURGY
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F3/1268—Membrane bioreactor systems
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- C—CHEMISTRY; METALLURGY
- 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
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- C—CHEMISTRY; METALLURGY
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- 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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- C—CHEMISTRY; METALLURGY
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
The invention relates to a Delftia sp for regulating quorum sensing quenching, a separation method and application thereof, wherein the Delftia sp JL5 strain is named as Delftia sp, the preservation unit is the China center for type culture Collection, the preservation time is 2021 year, 1 month and 21 days, the preservation number is CCTCCM2021139, and the address is as follows: wuhan university in Wuhan city, Hubei province. The dalfot bacteria related by the invention are bacteria for producing acylase, the enzyme expression quantity is very high, compared with the rhodococcus rhodochrous BH4 which is most widely applied at present, the dalfot bacteria show stronger N-Acyl Homoserine Lactone (AHL) degradation capability, and by virtue of the high-efficiency AHL degradation capability, when the dalfot bacteria are added into a Membrane Bioreactor (MBR), the attachment of a biological membrane on a membrane component can be inhibited, so that the pollution and blockage of the control membrane can be prevented, the water outlet efficiency is improved, and the operation cost is reduced.
Description
Technical Field
The invention belongs to the technical field of microbial separation, and particularly relates to a Delftia capable of efficiently quenching N-acyl homoserine lactone, a separation method and application thereof, and in particular relates to a Delftia capable of regulating quorum sensing quenching, a separation method and application thereof.
Background
Quorum sensing refers to the change in physiological and biochemical properties of a microbial population due to an increase in population density during its growth, which indicates characteristics that a small number of microbial cells or individual microbial cells do not possess. The reason for this change is that: when the microbial population density in the environment reaches a threshold value, the concentration of the signal molecules also reaches a certain level, and the signal transmission of related proteins including receptor proteins induces or inhibits the signal to be finally transmitted into cells, thereby influencing the expression of specific genes and regulating and controlling the physiological characteristics of microbial populations, such as bioluminescence, antibiotic synthesis, biofilm formation and the like.
Inter-species quorum-sensing quenching by substances produced by bacterial cells is an advanced technology that can be used for wastewater treatment, and is well suited for control of biofouling in Membrane Bioreactors (MBR). The main reason for MBR membrane fouling is that bacteria generate a biological membrane through quorum sensing to form membrane biological fouling, so that biological fouling can be effectively inhibited by inhibiting quorum sensing. The Quorum Quenching (QQ) technology mainly degrades and inactivates the AHL through N-Acyl Homoserine Lactone (AHL) quenching enzyme (namely, lactonase and acyltransferase) produced by bacteria, thereby reducing quorum induction intensity among bacteria, finally inhibiting the generation of a biological membrane on the membrane surface and achieving the purpose of controlling the pollution of an MBR membrane.
The common methods for preventing membrane fouling and blocking by MBR are mainly physical and chemical methods, such as back washing, chemical reagent adding and the like, but the physical and chemical methods are difficult to solve the fouling and blocking problem caused by organisms (bacteria form a compact biological membrane on the surface of the membrane). The current research is more favored to control from the biological point of view, and bacterial quorum sensing (bacterial communication mechanism) is the main cause of biological fouling, namely bacterial generation and bacterial fluxReceiving signal molecules, sensing the change of surrounding flora, and generating group behaviors (biomembrane generation and the like). AHL is ubiquitous in G-A class of signal molecules for bacteria whose concentration is reduced effectively inhibits their collective behavior can be achieved by bacteria that produce AHL degrading enzymes.
However, most of the prior art quenching bacteria capable of producing acylase have the defects of low expression level and low efficiency of acylase, and are not suitable for practical application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a Delftia capable of efficiently quenching N-acyl homoserine lactone and a separation method and application thereof, in particular to the Delftia capable of regulating quorum sensing quenching and the separation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a dalfot bacterium for regulating quorum sensing quenching, which is named as dalfot bacterium (Delftia sp.) JL5 strain, the preservation unit is the China center for type culture Collection, the preservation time is 21/1/2021, the preservation number is CCTCCM2021139, and the address is: wuhan university in Wuhan city, Hubei province.
The dalfot bacteria related by the invention are bacteria for producing acylase, the enzyme expression quantity is very high, compared with the rhodococcus rhodochrous BH4 which is most widely applied at present, the dalfot bacteria show stronger N-Acyl Homoserine Lactone (AHL) degradation capability, and by virtue of the high-efficiency AHL degradation capability, when the dalfot bacteria are added into a Membrane Bioreactor (MBR), the attachment of a biological membrane on a membrane component can be inhibited, so that the pollution and blockage of the control membrane can be prevented, the water outlet efficiency is improved, and the operation cost is reduced.
In a second aspect, the present invention provides a method for isolating the dalfot bacterium that regulates quorum sensing quenching according to the first aspect, wherein the method for isolating the dalfot bacterium comprises:
(1) transferring an activated sludge sample in the membrane bioreactor to a culture medium taking N-acyl homoserine lactone as a unique carbon source for enrichment culture, and then coating the activated sludge sample on a flat plate for purification to form a single colony;
(2) selecting a single colony with complete morphology for comparison of N-acyl homoserine lactone quenching capacity, and screening out a high-efficiency quenching strain;
(3) and performing morphological, physiological, biochemical and molecular biological analysis on the screened quenching bacterial strain, determining species information, and identifying to obtain the Delftia bacterium JL5 bacterial strain for regulating quorum sensing quenching.
Preferably, the medium containing N-acylhomoserine lactone as a sole carbon source in step (1) comprises Na2HPO4、KH2PO4、NaCl、NH4Cl、MgSO4、CaCl2Trace elements and N-acyl homoserine lactones.
Preferably, the medium containing N-acylhomoserine lactone as a sole carbon source in step (1) comprises Na2HPO4 4-8％、KH2PO4 1-5％、NaCl 0.2-0.8％、NH4Cl 0.5-2％、MgSO4 0.5-2mM、CaCl20.5-2 μ M, 0.5-2% trace elements and 1-3mM N-acyl homoserine lactone.
The Na is2HPO4The content of (b) may be 4%, 5%, 6%, 7%, 8%, etc.
The KH2PO4The content of (b) may be 1%, 2%, 3%, 4%, 5%, etc.
The mass percentage of the NaCl can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% and the like.
The NH4The mass percentage of Cl may be 0.5%, 1%, 1.5%, 2%, etc.
The MgSO4The concentration of (B) may be 0.5mM, 1mM, 1.5mM, 2mM, or the like.
The CaCl is2The concentration of (b) may be 0.5. mu.M, 1. mu.M, 1.5. mu.M, 2. mu.M, etc.
The mass percentage of the trace elements can be 0.5%, 1%, 1.5%, 2% and the like.
The concentration of the N-acyl homoserine lactone may be 1mM, 1.5mM, 2mM, 3mM, or the like.
Other specific values in the above ranges can be selected, and are not described in detail herein.
Preferably, the N-acyl homoserine lactone is N-hexanoyl homoserine lactone (C6-HSL).
Preferably, the enrichment culture of step (1) is performed for 18-30h (e.g., 18h, 20h, 24h, 28h, 30h, etc.) at a temperature of 28-32 deg.C (e.g., 28 deg.C, 29 deg.C, 30 deg.C, 31 deg.C, 32 deg.C, etc.), 100-300r/min (e.g., 100r/min, 150r/min, 200r/min, 250r/min, 300r/min, etc.) with shaking. Other specific values in the above ranges can be selected, and are not described in detail herein.
Preferably, before the activated sludge sample in the membrane bioreactor is transferred in the step (1), the sample is diluted by ultrapure water and inoculated into a culture medium for culture.
Preferably, the inoculation amount is 3-7% (v/v), such as 3%, 4%, 5%, 6%, 7%, etc., and other specific values within the range of the values can be selected, which is not described herein again.
Preferably, the culturing condition is carried out under the shaking condition of temperature of 28-32 ℃ (for example, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃ and the like), 100-300r/min (for example, 100r/min, 150r/min, 200r/min, 250r/min, 300r/min and the like) for 40-56h (for example, 40h, 42h, 45h, 48h, 50h, 56h and the like), and other specific values in the above numerical value ranges can be selected, so that the detailed description is omitted.
Preferably, the transfer amount is 1-3% (v/v), such as 1%, 2%, 3%, etc., and other specific values in the value range can be selected, which is not described herein again.
Preferably, the comparison method of quenching ability of N-acyl homoserine lactone in step (2) is: carrying out mixed culture on the single bacterial colony and the N-acyl homoserine lactone in a culture medium, centrifuging, collecting supernatant, and inactivating; then co-culturing with a report strain in a culture medium, centrifuging to collect supernatant, adding dimethyl sulfoxide, ultrasonically breaking cells, centrifuging to collect supernatant, measuring OD value, calculating the concentration of N-acyl homoserine lactone in a sample according to a standard curve, and further comparing the quenching capacity of the N-acyl homoserine lactone.
The construction method of the standard curve may exemplarily be: samples containing 10, 5, 2.5, 1.25. mu. mol/L of N-acyl homoserine lactone such as C6-HSL were added to 10. mu.L to 1mL of the same bacterial suspension of the reporter strain, respectively, cultured under the same culture conditions as described above, centrifuged to collect the supernatant, added with dimethyl sulfoxide, cells were disrupted by sonication, centrifuged to collect the supernatant, and OD value was measured to plot a standard curve.
In a fourth aspect, the invention provides a use of the dalfot bacterium that regulates quorum sensing quenching as described in the first aspect for inhibiting and/or inhibiting bacterial biofilm.
The MBR membrane fouling is mainly caused by that bacteria generate a biological membrane through quorum sensing to form membrane biological fouling, and the Delftia bacterium JL5 strain related by the invention degrades and inactivates an N-Acyl Homoserine Lactone (AHL) produced by quenching enzyme (namely acyltransferase), thereby reducing quorum sensing intensity among bacteria and finally inhibiting the generation of the biological membrane on the membrane surface.
In a fifth aspect, the invention provides an application of the dalfot bacteria for regulating and controlling quorum sensing quenching in membrane bioreactor for preventing membrane fouling.
When the Delftia bacterium JL5 strain is applied to a membrane bioreactor, N-Acyl Homoserine Lactone (AHL) produced quenches enzyme (namely acyltransferase), so that AHL is degraded and inactivated, thereby reducing the quorum sensing intensity among bacteria, finally inhibiting the generation of a biological membrane on the surface of the membrane and achieving the purpose of controlling the pollution of an MBR membrane.
Compared with the prior art, the invention has the following beneficial effects:
the dalfot bacteria related by the invention are bacteria for producing acylase, the enzyme expression quantity is very high, compared with the rhodococcus rhodochrous BH4 which is most widely applied at present, the dalfot bacteria show stronger N-Acyl Homoserine Lactone (AHL) degradation capability, and by virtue of the high-efficiency AHL degradation capability, when the dalfot bacteria are added into a Membrane Bioreactor (MBR), the attachment of a biological membrane on a membrane component can be inhibited, so that the pollution and blockage of the control membrane can be prevented, the water outlet efficiency is improved, and the operation cost is reduced.
Drawings
FIG. 1 is a graph showing the results of quenching ability of each candidate strain to C6-HSL;
FIG. 2 is a gram stain of Delftia JL 5;
the bacterial strain JL5 of Delftia sp, the preservation unit is China center for type culture Collection, the preservation time is 1 month and 21 days 2021 year, the preservation number is CCTCCM2021139, the address is: wuhan university in Wuhan city, Hubei province;
FIG. 3 is a scanning electron microscope photograph of bacterium Delftia JL 5;
FIG. 4 is a graph of the biofilm formation and biofilm formation quantitative statistics in single and double biofilm groups;
FIG. 5 is a graph showing the results of an AHL delinking recovery test.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Pseudomonas aeruginosa PAO1(ATCC 15692) referred to in the examples below was derived from ATCC strain stock; rhodococcus sp.BH4(KCTC 33122) and Agrobacterium tumefaciens A136(ATCC 51350) were gifts from professor Licheng school of chemistry and bioengineering, Seoul university of national university; the reporter strain Chromobacterium violacea VIR24(ATCC 12472) was derived from the ATCC strain pool.
Example 1
The embodiment provides a dalfot bacterium JL5 strain for regulating quorum sensing quenching, and the separation method comprises the following steps:
(1) taking an activated sludge sample in MBR, diluting the activated sludge sample by 10 times with ultrapure water, inoculating the sample into LB culture medium by 5% (v/v), carrying out shake culture at the constant temperature of 30 ℃ for 48h at 200r/min, transferring the culture solution into a basic culture medium with 2% (v/v) of 2mmol/L C6-HSL as a unique carbon source (the formula is shown in Table 1, and the formula of 1000 x trace elements in the formula is shown in Table 2), and carrying out shake enrichment culture at the constant temperature of 30 ℃ for 24h at 200 r/min.
TABLE 1
TABLE 2
(2) Diluting the culture solution obtained in the step (1) by 100 times with sterile water, spreading the diluted culture solution on an LB agar plate, culturing at a constant temperature of 30 ℃ for 48 hours, and selecting colonies with different morphologies for separation and purification.
(3) Respectively inoculating single colonies with complete forms into LB culture medium, performing shake culture at 30 ℃ for 24h at 200r/min, diluting with fresh LB until OD600 is 1.0, adding 10 mu mol/L C6-HSL, performing shake culture at 30 ℃ at 200r/min, respectively taking 500 mu L of culture solution at 5h and 10h, centrifuging at 8000r/min for 5min, collecting supernatant, inactivating the supernatant in a constant-temperature water bath kettle at 95 ℃ for 5min, cooling, and performing freeze preservation at-20 ℃.
(4) Reporter strain VIR24 was cultured in LB medium at 28 ℃ for 24h, and the broth was diluted to OD600 of 0.1 for use. Standard samples (from C) containing 10, 5, 2.5, 1.25, 0.625. mu. mol/L C6-HSL10H17NO3(C6-HSL, M.W 199.25, CAS: 147852-83-3) samples were prepared with water, purchased from Sigma under the accession number 56695), and 10. mu.L to 1mL of the above VIR24 bacterial solution were added, and cultured with shaking at 200r/min at 30 ℃ for 24 hours. 8000r/min, centrifuging for 5min, taking supernatant, adding 150 mu L dimethyl sulfoxide (DMSO) for resuspension, breaking ultrasonic cells for 10min, centrifuging again, taking 100 mu L supernatant, adding into a 96-well plate, measuring the light absorption value of a sample at 530nm, and constructing a standard curve.
(5) And (4) respectively adding 10 mu L to 1mL of the VIR24 bacterial liquid into the sample obtained in the step (3), repeating the step in the step (4), measuring the light absorption value of the sample at 530nm, calculating the content of C6-HSL in the sample according to a standard curve, and screening the strain with the strongest AHL quenching capacity. The degradation of C6-HSL at 5h and 10h for each isolate is shown in FIG. 1. As can be seen from fig. 1: after 5h of co-culture with C6-HSL, JL5 reduced C6-HSL from the initial 10. mu. mol/L to 1.2. mu. mol/L with a degradation rate of 88%, while other isolates SZU83, SZU18, XM19, FF19 and SZU68 degraded C6-HSL by about 40% -50%; after 10h of culture, JL5 almost completely degrades C6-HSL, SZU18 and SZU68 in the system to reach 70%; SZU83, XM19 and FF19 were degraded at 60%, 58% and 68%, respectively, and were less decomposed than JL5, SZU18 and SZU68 with respect to C6-HSL. In the control sample without any addition of the strain C6-HSL, the content of C6-HSL is slightly reduced after 10 hours of culture, and the natural degradation rate of the signal molecule is about 10%.
The above results show that JL5 has strong AHL signal molecule degradation ability (C6-HSL in the culture solution is basically and completely degraded at 10 h).
(6) And (3) performing morphological, physiological, biochemical and molecular biological analysis on the screened strains respectively, determining species information, and identifying to obtain the Delftia bacterium JL5 strain for regulating quorum sensing quenching.
Example 2
The strain selected in example 1 was morphologically observed, gram-stained and observed under a microscope, as shown in FIG. 2, from which: the bacterial colony on the LB agar plate has regular edges and smooth surface, is an opaque yellowish bacterial colony and is negative in gram stain.
When the specimen was observed by a scanning electron microscope, as shown in FIG. 3, it can be seen that: the strain is rod-shaped, the length of the strain is about 2.6-3.1 μm, and the width of the strain is about 0.6-0.8 μm.
Example 3
The strain obtained by screening in example 1 is subjected to a physiological and biochemical characteristic identification experiment, and the result shows that: the glucose oxidation fermentation test, the citrate test and the catalase test are positive; M.R test, indole test, V.P test, starch hydrolysis test, gelatin hydrolysis test, and acetyl methyl methanol test were negative.
Example 4
The strains screened in example 1 were subjected to 16S rDNA molecular biology identification. Bacterial genomic DNA was extracted using an Ezup column type bacterial genomic DNA extraction kit (Shanghai, Ltd.), and the 16S rDNA gene of the bacterium was amplified using a pair of universal primers 27F (5-AGATTTGATCCTGGCTCAG-3) and 1492R (5-GGTTACCTTGTTACGACTT-3). And purifying PCR products and sequencing (Shanghai worker), wherein the sequencing result is subjected to sequence identification through BLAST of NCBI and then is compared with sequences in a GenBank database to obtain related species information. The results showed 99.93% homology to Delftia lactisteri of the genus Delftia, which was identified as Delftia sp.
The 16S rDNA base sequence is as follows:
TGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGTAACAGGTCTTCGGACGCTGACGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAGTCGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATACGATCTGAGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGGGATAAAAGCTTACCAAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAAAAGCTCCTTCTAATACAGGGGGCCCATGACGGTACCGTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGTGACTGCATGGCTAGAGTACGGTAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGACCTGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAATTAGTTTTCTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTTGACATGGCAGGAAGTTTCCAGAGATGGATTCGTGCTCGAAAGAGAACCTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACATTCAGTTGAGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTCTCGCCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTTACCACGGCGGGGTTCGTGACTGGGGTGAAGTCGTA。
according to the results of the physiological and biochemical characteristics, the colony morphological characteristics, the individual morphological characteristics and the molecular biology identification result are combined, and the bacterium can be finally determined to be the Delftia sp.
Example 5
In this example, the bacterial biofilm inhibiting ability of strain JL5 was evaluated as follows:
the influence of the strain JL5 on the formation of a pseudomonas aeruginosa biofilm is evaluated through the species interaction of the strain JL5 and QS bacteria in the biofilm development process. Before the experiment, the strain was cultured in 10mL of LB medium at 30 ℃ for 24h at 200 rpm. Cell density was quantified spectrophotometrically and diluted to OD600Is 0.05 for standby.
The test was divided into a single bacterial biofilm group (i.e. biofilm formation when JL5 bacteria alone and Pseudomonas aeruginosa alone were cultured): adding 900 μ L LB culture medium and 100 μ L bacterial dilution into 24-well plate; and a double-bacterial biofilm group (namely biofilm formation of the single JL5 bacterium and pseudomonas aeruginosa in mixed culture): to a 24-well plate, 900. mu.L of LB medium and 50. mu.L of JL5 strain dilutions were added, as well as 50. mu.L of P.aeruginosa dilutions. Sterile LB medium was used as a negative control.
The biofilm grew at 30 ℃ without agitation. After 24h, the cells were removed and washed 2 times with PBS buffer. The biofilm was stained with 1500mL of 0.1% crystal violet solution per well for 15 min. Then rinsed 2 times with deionized water and air dried. The biofilm layer was dissolved in 1500mL of 30% acetic acid per well and incubated at 25 ℃ for 15 min. The absorbance was measured at 550 nm. The results are shown in FIG. 4, and it can be seen from FIG. 4 that: JL5 is weak in biofilm formation capability, PAO1 has strong biofilm formation capability in single culture, and JL5 reduces the biofilm formation amount of PAO1 by 57%, which shows that JL5 has obvious inhibition capability on biofilm formation of PAO 1.
Example 6
In this example, strain JL5 was tested for AHL chain scission recovery by the following procedure:
AHL is degraded by lactonase and acylase, but AHL lactonase is hydrolyzed and then reversely reacts under specific conditions (acidity), so that lactonase-producing bacteria may have a problem of low efficiency in actual operation, and most of the studies are now carried out under the class of lactonase, and in order to confirm that the strain JL5 of the present invention is an acyltransferase-producing bacterium, the following experiment was carried out.
The isolated strain JL5 was inoculated into 30mL of LB medium and cultured at 30 ℃ and 200rpm for 24 hours, followed by centrifugation at 4 ℃ and 6000rpm for 10 min. The supernatant was filtered using a 0.22 μm needle filter and the pellet resuspended in an equal volume of Tris-Cl buffer (50mM, pH 7.0). N-octanoylhomoserine lactones (C8-HSL, available from Sigma, M.W: 227.30, CAS: 147852-84-4, cat # 44558) (200nM) were then added, respectively, and sampling was performed at different times (1h, 2h, 3h, 4 h).
mu.L of each of the above samples was mixed with 20. mu.L of 1M HCl, incubated at 4 ℃ for 48 hours, then neutralized by adding 40. mu.L of 1M phosphate buffer (pH 7) to each sample, and then 10. mu.L of the mixture was applied to A136 agar plates for observation. When a sample degraded with Rhodococcus BH 4(a lactonase-producing quenching bacterium) was used as a control, the results are shown in FIG. 5, in JL5, the C8-HSL structure was not restored, and the acyl chain was irreversibly cleaved; BH4 is a QQ bacterium known to produce lactamase and has been detected as C8-HSL in degradation repair experiments. It was shown that the enzymes produced by these two QQ bacteria were different (lactamase and acyltransferase, respectively), and therefore strain JL5 was an acylase-producing strain, while the study of the enzyme type produced by the genus Delftia belonging to acylases in Maisuria et al (Maisuri et al, 2015) was also verified.
The applicant states that the present invention is described by the above examples to a dalbergia regulating quorum sensing quenching and the isolation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. The Delftia sp regulating quorum sensing quenching is named as Delftia sp JL5 strain, the preservation unit is the China center for type culture Collection, the preservation time is 2021 year, 1 month and 21 day, the preservation number is CCTCCM2021139, and the address is as follows: wuhan university in Wuhan city, Hubei province.
2. The method for isolating a dalbergia regulating quorum sensing quenching according to claim 1, wherein the isolation method comprises:
(1) transferring an activated sludge sample in the membrane bioreactor to a culture medium taking N-acyl homoserine lactone as a unique carbon source for enrichment culture, and then coating the activated sludge sample on a flat plate for purification to form a single colony;
(2) selecting a single colony with complete morphology for comparison of N-acyl homoserine lactone quenching capacity, and screening out a high-efficiency quenching strain;
(3) and performing morphological, physiological, biochemical and molecular biological analysis on the screened quenching bacterial strain, determining species information, and identifying to obtain the Delftia bacterium JL5 bacterial strain for regulating quorum sensing quenching.
3. The method for isolating Delftia bacterium that regulates quorum sensing quenching according to claim 2, wherein the medium containing N-acylhomoserine lactone as a sole carbon source in the step (1) comprises Na2HPO4、KH2PO4、NaCl、NH4Cl、MgSO4、CaCl2Trace elements and N-acyl homoserine lactones;
preferably, the medium containing N-acylhomoserine lactone as a sole carbon source in step (1) comprises Na2HPO4 4-8％、KH2PO4 1-5％、NaCl 0.2-0.8％、NH4Cl 0.5-2％、MgSO4 0.5-2mM、CaCl20.5-2 μ M, 0.5-2% trace elements and 1-3mM N-acyl homoserine lactone.
4. The method for isolating Delftia bacterium that regulates quorum sensing quenching according to claim 3, wherein the N-acyl homoserine lactone is N-hexanoyl homoserine lactone.
5. The method for separating Delftia bacterium which regulates quorum sensing quenching as claimed in any one of claims 2 to 4, wherein the enrichment culture of step (1) is performed at a temperature of 28-32 ℃ under the shaking condition of 100-300r/min for 18-30 h.
6. The method for separating Delftia from regulated quorum sensing quenching according to any one of claims 2 to 5, wherein the activated sludge sample in the membrane bioreactor is diluted with ultrapure water and inoculated into a culture medium for culture before being transferred in the step (1).
7. The method for isolating Delftia bacterium that regulates quorum sensing quenching according to claim 6, wherein the inoculation amount is 3 to 7% (v/v);
preferably, the culturing is carried out for 40-56h under the conditions of temperature of 28-32 ℃ and shaking at the speed of 100-300 r/min;
preferably, the amount of transfer is 1-3% (v/v).
8. The method for isolating Delftia bacterium that regulates quorum sensing quenching according to any one of claims 2 to 7, wherein the N-acyl homoserine lactone quenching ability comparison method in the step (2) comprises: carrying out mixed culture on the single bacterial colony and the N-acyl homoserine lactone in a culture medium, centrifuging, collecting supernatant, and inactivating; then co-culturing with a report strain in a culture medium, centrifuging to collect supernatant, adding dimethyl sulfoxide, ultrasonically breaking cells, centrifuging to collect supernatant, measuring OD value, calculating the concentration of N-acyl homoserine lactone in a sample according to a standard curve, and further comparing the quenching capacity of the N-acyl homoserine lactone.
9. Use of the daloft bacterium which regulates quorum sensing quenching according to claim 1 for inhibiting and/or inhibiting bacterial biofilm.
10. The use of the dalfot bacteria for regulating quorum sensing quenching as defined in claim 1 in membrane bioreactor for preventing membrane fouling.
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