CN111499601B - Preparation method and application of AHL signal molecule - Google Patents

Preparation method and application of AHL signal molecule Download PDF

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CN111499601B
CN111499601B CN202010212934.7A CN202010212934A CN111499601B CN 111499601 B CN111499601 B CN 111499601B CN 202010212934 A CN202010212934 A CN 202010212934A CN 111499601 B CN111499601 B CN 111499601B
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hsl
slant
dichloromethane
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CN111499601A (en
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陈东之
刘浩阳
王双
王莉宁
陈建孟
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Zhejiang Ocean University ZJOU
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Abstract

The invention provides a preparation method and application of AHL signal molecules, which belong to the technical field of environmental protection, and comprise the steps of preparing an AHL signal molecule production source by taking Methylobacterium rhodesianum H13 as a strain, and extracting 3OC from the AHL signal molecule production source 8 HSL, 3OC addition with exogenous addition 8 Mode of HSL, enhancement of the degradation of dichloromethane to CO by strain Methylobacterium rhodesianum H13 2 And H 2 O. Under pure culture condition, the strain is exogenously added with 3OC 8 The HSL has better degradation capability to dichloromethane in the 800-2400 nM range; exogenous addition of 3OC 8 The HSL can obviously accelerate the growth of a film on the filler of the strain, thereby providing a powerful guarantee for engineering application of purifying dichloromethane by a biological method.

Description

Preparation method and application of AHL signal molecule
Technical Field
The invention belongs to the technical field of environmental protection, and particularly relates to a preparation method and application of an AHL signal molecule.
Background
Dichloromethane (DCM) is an important organic solvent and intermediate product and is widely used in many industries. However, it has a low boiling point and is easily volatilized into the atmosphere, causing atmospheric pollution. DCM has a high solubility in lipids, is inhaled by humans or animals, is easily accumulated in adipose tissues of organisms, and often has three causes of carcinogenesis, teratogenicity and mutagenesis, and is one of 129 toxic pollutants preferentially controlled by the us environmental protection agency. Therefore, the method has important social and environmental significance for effectively removing the DCM.
After many years of efforts, many researchers have isolated pure bacterial strains from environmental media using DCM as the only carbon and energy source, and we have also isolated and purified in 2010 a laboratory to obtain methylene chloride-degrading bacteria, methylene robusta (Methylobacterium rhodesianum) H13, which was deposited in the chinese collection of typical cultures. In subsequent applications, the biotrickling filter inoculated with the M.rhodesian H13 strain achieved a dichloromethane removal efficiency of 80-99% but a film formation period of up to 30 days. This demonstrates how effective control of biofilm on packing has become one of the key means to strengthen trickling filtration towers to remove methylene chloride. The biological filler is the core part of the biological trickling filtration tower, and the biological film on the filler in the tower can effectively degrade organic pollutants, so that the film hanging of the filler is a key step of the biological purification process. At present, the biofilm formation time of the biological trickling filtration process is generally long, and the biofilm formation is influenced by multiple factors including operating conditions, nutritional ingredients, microorganism types, filler properties and the like. More and more studies in recent years have shown that quorum sensing QS (quorum sensing) has a close correlation with bacterial biofilm formation, in which quorum sensing AHL signaling molecules play a key role.
The prior art, such as the Chinese patent publication No. CN 101993839B, discloses a strain capable of degrading DCM, namely, methylobacillus roteierides (Methylobacillus) H13, which is preserved in China center for type culture Collection, address: china, university of Wuhan, 430072, date of preservation: 20 days 5 months 2010, deposit number: cctccc No: m2010121. The strain is aerobic gram-negative bacillus, can grow by taking DCM as the only carbon source and energy source and simultaneously efficiently degrade the substrate, and lays a foundation for engineering application of purifying DCM-containing wastewater and waste gas efficiently by a biological method.
Disclosure of Invention
The invention aims at providing an AHL signal molecule-N-3-oxo Xin Xiangao serine lactone (3 OC) generated by Methylobacterium rhodesianum H13 8 -HSL) and its use in enhancing microbial degradation of methylene chloride, 3OC is enhanced by optimizing the preparation process 8 HSL production and by control of 3OC 8 Quantity of HSLThe biomembrane on the filler is regulated and controlled, so that the film forming starting period of the process is shortened, and the removal of dichloromethane is enhanced.
The technical scheme adopted by the invention for achieving the purpose is as follows:
providing a population induction AHL signal molecule, wherein the Chinese name is N-3-oxo Xin Xiangao serine lactone, and the chemical formula is 3OC 8 -HSL, said quorum sensing AHL signaling molecule being produced by a dichloromethane degrading bacterium.
Preferably, the methylene chloride-degrading bacterium is Methylobacillus robustus (Methylobacterium rhodesianum) H13.
The preparation method of the population induction AHL signal molecule comprises the following steps:
a. slant culture: inoculating Methylobacterium rhodesianum H to a slant LB solid culture medium, and culturing at 30 ℃ for 24-36 h to obtain slant thalli;
b. and (3) performing expansion culture: inoculating the slant thallus obtained in the step a into LB liquid medium by using an inoculating loop, culturing at 30 ℃ for 24-36 h to obtain OD 600 Bacterial liquid of 0.6-0.8;
c. c, taking the bacterial liquid in the step b, centrifuging at 1000rpm for 15min, taking supernatant, filtering, extracting the filtrate with ethyl acetate in equal volume for three times, combining the extracts, evaporating the solvent by nitrogen blowing, concentrating, and storing the extract at-80 ℃ for later use;
the final concentration composition of LB solid medium in step a is: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;
the final concentration composition of LB liquid medium in step b is: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
Preferably, OD 600 ＝0.7。
Provides the use of a population-sensing AHL signal molecule in enhancing microbial degradation of methylene dichloride.
A method for enhancing microbial degradation of methylene chloride is provided, comprising: dichloromethane is used as a substrate, methylobacterium rhodesianum H13 is used as degradation bacteria, an inorganic salt culture medium is used as a medium, and 3OC is added 8 -HSL degrading dichloromethane at ph=5.5-10, rotation speed 160rpm, temperature 10-40 ℃.
More preferably, the ph=6.5 to 7.5 and the temperature is 20 to 35 ℃.
Preferably, the initial concentration of the dichloromethane is 50-1000mg/L,3OC 8 The exogenous addition amount of the HSL is 0-2400nM.
More preferably, the initial concentration of the methylene chloride is 300-500mg/L.
More preferably, the 3OC 8 The exogenous addition amount of the HSL is 800-1600nM.
Preferably, the 3OC is as described above 8 The preparation method of the HSL comprises the following steps: methylobacterium rhodesianum H13 is used as a strain to prepare an AHL signal molecule production source, and 3OC is extracted from the AHL signal molecule production source 8 -HSL; the AHL signal molecule production source is OD with inorganic salt culture medium as culture medium 600 0.6-0.8 M.rhodesian H13 bacterial liquid.
More preferably, the AHL signal molecule source is 1L OD using inorganic salt culture medium as culture medium 600 0.7 of Methylobacterium rhodesianum H13.
Preferably, the methylene chloride is in a gaseous state.
More preferably, the concentration of the methylene chloride gas is 500mg/m 3 。
Preferably, the preparation method of the AHL signal molecule production source specifically comprises the following steps:
slant culture: inoculating Methylobacterium rhodesianum H to a slant LB solid culture medium, and culturing at 30 ℃ for 24-36 h to obtain slant thalli;
and (3) performing expansion culture: inoculating the slant thallus obtained in the step 1) into LB liquid medium by using an inoculating loop, and culturing for 24-36 h at 30 ℃ to obtain OD 600 Bacterial liquid of 0.6-0.8;
the final concentration composition of the LB solid medium is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;
the final concentration composition of the LB liquid medium is as follows: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
Preferably, OD 600 ＝0.7。
The beneficial effects of the invention are as follows:
the invention provides an AHL signal molecule (3 OC) generated by Methylobacterium rhodesianum H13 8 -HSL), by exogenously adding 3OC 8 The HSL can promote the growth of Methylobacterium rhodesianum H13, is favorable for rapid biofilm mounting of biological fillers, strengthens the biodegradation of dichloromethane, and provides a new technical choice for engineering application of purifying the dichloromethane-containing wastewater and waste gas by a biological method.
Drawings
FIG. 1 is a graph showing the detection results of AHL signal molecules in example 1 of the present invention;
FIG. 2 is a schematic diagram of a different 3OC in example 2 of the present invention 8 -bar graph of the effect of HSL exogenous addition on dichloromethane degradation (a), growth (B), mineralization (C) of Methylobacterium rhodesianum H;
FIG. 3 is a measurement result of SAM concentration in test example 1 according to the present invention;
FIG. 4 is a 3OC in test example 1 of the present invention 8 -determination of HSL content;
FIG. 5 is a measurement result of polysaccharide and protein content in test example 2 of the present invention;
FIG. 6 is a graph showing the results of measuring the biological membrane biomass in test example 2 according to the present invention;
FIG. 7 is a measurement result of the activity of a biological membrane dehydrogenase per unit mass in test example 2 of the present invention;
FIG. 8 shows the results of measuring the removal rate of methylene chloride off-gas in test example 3 according to the present invention.
Detailed Description
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety unless otherwise indicated, as if set forth in full.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, definitions, will control.
When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a series of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is described, the described range should be interpreted as including a range of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. Where numerical ranges are described herein, unless otherwise stated, the ranges are intended to include the range endpoints and all integers and fractions within the range.
In addition, the words "a" and "an" preceding an element or component of the present invention are intended to mean that there is no limitation on the number of times that the element or component occurs. Thus, the use of "a" or "an" is to be understood as including one or at least one of the elements or components in the singular, unless the amount is explicitly stated as being a single number.
Embodiments of the invention, including the embodiments of the invention described in the summary section and any other embodiments described herein below, may be arbitrarily combined.
The present invention is described in detail below.
The technical scheme adopted by the invention for achieving the purpose is as follows:
providing a population induction AHL signal molecule, wherein the Chinese name is N-3-oxo Xin Xiangao serine lactone, and the chemical formula is 3OC 8 -HSL, said quorum sensing AHL signaling molecule being produced by a dichloromethane degrading bacterium.
Preferably, the methylene chloride-degrading bacterium is Methylobacillus robustus (Methylobacterium rhodesianum) H13.
The preparation method of the population induction AHL signal molecule comprises the following steps:
a. slant culture: inoculating Methylobacterium rhodesianum H to a slant LB solid culture medium, and culturing at 30 ℃ for 24-36 h to obtain slant thalli;
b. and (3) performing expansion culture: inoculating the slant thallus obtained in the step a into LB liquid medium by using an inoculating loop, culturing at 30 ℃ for 24-36 h to obtain OD 600 Bacterial liquid of 0.6-0.8;
c. c, taking the bacterial liquid in the step b, centrifuging at 1000rpm for 15min, taking supernatant, filtering, extracting the filtrate with ethyl acetate in equal volume for three times, combining the extracts, evaporating the solvent by nitrogen blowing, concentrating, and storing the extract at-80 ℃ for later use;
the final concentration composition of LB solid medium in step a is: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;
the final concentration composition of LB liquid medium in step b is: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
Preferably, 0.024-0.036 g/L4-hydroxy-3-methoxybenzyl alcohol is added to the LB liquid medium in the step b. The chemical synthesis of the gram-negative bacterial (G-) signal molecule AHL by intracellular related enzymes requires the provision of homoserine lactones from acyl donor (acyl-ACP) and ademetionine (SAM) as substrates. 4-hydroxy-3-methoxyl benzyl alcohol can promote Methylobacterium rhodesianum H to synthesize SAM, promote SAM to react with acyl donor to synthesize homoserine lactone, and promote 3OC 8 Synthesis of HSL such that 3OC 8 The process for the preparation of HSL has the advantage of low cost and high yields.
Preferably, OD 600 ＝0.7。
Provides the use of a population-sensing AHL signal molecule in enhancing microbial degradation of methylene dichloride.
A method for enhancing microbial degradation of methylene chloride is provided, comprising: dichloromethane is used as a substrate, methylobacterium rhodesianum H13 is used as degradation bacteria, an inorganic salt culture medium is used as a medium, and 3OC is added 8 -HSL degrading dichloromethane at ph=5.5-10, rotation speed 160rpm, temperature 10-40 ℃.
Preferably, the inorganic salt culture medium is additionally provided with (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene, 3OC 8 The molar ratio of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene to HSL is 4.2-5.3:1. As biomass increases, the amount of EPS secreted by the bacteria changes accordingly, and when the EPS amount accumulates to a certain concentration, the suspended microorganisms will switch to an attached state. Initially the bacteria are immobilized on the surface of the carrier by means of a gel linkage, and as the bacteria grow, the bacteria between different populations and the same population are linked mainly by secretion of EPS. Therefore EPS production is one of the properties of biofilms and it is of paramount importance to study the role of EPS in biofilm formation. 3OC 8 The molar ratio of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene to 3OC is 4.2-5.3:1 8 The synergistic effect of the HSL can promote the secretion of EPS by cells, improve the connection effect between bacteria, facilitate the formation of a biological film, increase the amount of the biological film, reduce the film forming starting time, and improve the metabolic activity of Methylobacterium rhodesianum H13 cells, thereby further strengthening the degradation capability of dichloromethane.
More preferably, the ph=6.5 to 7.5 and the temperature is 20 to 35 ℃.
Preferably, the initial concentration of the dichloromethane is 50-1000mg/L,3OC 8 The exogenous addition amount of the HSL is 0-2400nM.
More preferably, the initial concentration of the methylene chloride is 300-500mg/L.
More preferably, the 3OC 8 The exogenous addition amount of the HSL is 800-1600nM.
Preferably, the 3OC is as described above 8 The preparation method of the HSL comprises the following steps: methylobacterium rhodesianum H13 is used as a strain to prepare an AHL signal molecule production source, and 3OC is extracted from the AHL signal molecule production source 8 -HSL; the AHL signal molecule production source is OD with inorganic salt culture medium as culture medium 600 0.6-0.8 of Methylobacterium rhodesianum H13 bacterial liquid.
More preferably, the AHL signal molecule is produced by inorganic salt culture1L OD of culture medium 600 0.7 of Methylobacterium rhodesianum H13.
Preferably, the methylene chloride is in a gaseous state.
More preferably, the concentration of the methylene chloride gas is 500mg/m 3 。
Preferably, the preparation method of the AHL signal molecule production source specifically comprises the following steps:
slant culture: inoculating Methylobacterium rhodesianum H to a slant LB solid culture medium, and culturing at 30 ℃ for 24-36 h to obtain slant thalli;
and (3) performing expansion culture: inoculating the slant thallus obtained in the step 1) into LB liquid medium by using an inoculating loop, and culturing for 24-36 h at 30 ℃ to obtain OD 600 Bacterial liquid of 0.6-0.8;
the final concentration composition of the LB solid medium is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;
the final concentration composition of the LB liquid medium is as follows: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
Preferably, OD 600 ＝0.7。
The invention is described in further detail below with reference to examples:
the composition of the mineral salts medium used in the examples is: na (Na) 2 HPO 4 ·12H 2 O 4.5g/L、KH 2 PO 4 1.0g/L、(NH 4 ) 2 SO 4 2.5g/L、MgSO 4 ·7H 2 O0.2 g/L, anhydrous CaCl 2 0.023g/L, 1mL/L of trace element mother liquor, pH7.0, and deionized water as solvent; the concentration of the trace element mother solution comprises the following components: feSO 4 ·7H 2 O 1.0g/L、CuSO 4 ·5H 2 O 0.02g/L、H 3 BO 3 0.014g/L、MnSO 4 ·4H 2 O 0.10g/L、ZnSO 4 ·7H 2 O 0.10g/L、Na 2 MoO 4 ·2H 2 O 0.02g/L、CoCl 2 ·6H 2 O0.02 g/L, and deionized water as solvent.
The composition of the LB solid medium used was: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, 18g/L agar, deionized water as solvent and natural pH value.
The composition of the LB liquid culture medium is as follows: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
The room temperature according to the invention is 26 ℃.
Example 1:
the preparation method of the population induction AHL signal molecule comprises the following steps:
a. slant culture: methylobacterium rhodesianum H13 (M.rhodesian H13) is inoculated in a slant LB solid culture medium and cultured for 30 hours at the temperature of 30 ℃ to obtain slant thalli;
b. and (3) performing expansion culture: inoculating the slant thallus obtained in step a into 1L liquid LB culture medium with inoculating loop, shake culturing at 30deg.C under 160r/min for 30 hr to obtain OD 600 Bacterial liquid=0.7;
c. extraction and concentration of AHL signal molecules: and c, taking the bacterial liquid in the step b, centrifuging at 1000rpm for 15min, taking supernatant, filtering, extracting the filtrate with ethyl acetate in equal volume for three times, combining the extracts, concentrating the nitrogen-blown volatile solvent to 10mL, keeping the AHL signal molecular content at 20mg/L, and keeping the extract at the temperature of-80 ℃ for later use.
Identification of AHL Signal molecules: and (3) qualitatively analyzing AHL signal molecules by adopting a gas chromatograph-mass spectrometer (GC-MS). The gas phase system is HP6890GC, an HP-5MS capillary column (30m*0.25mm ID*0.25um) is equipped, the carrier gas is helium (99.999%), the carrier gas flow rate is 0.8mL/min, the sample inlet temperature is 200 ℃, the split ratio is 150:1, and the auxiliary channel temperature is 280 ℃. Mass spectrometry conditions: ion source (70eV,500uA,MS Quad 150 ℃, MS source 230 ℃). Full scan mode: m/z=15-800, selecting ion scan mode: m/z=143. The detection result of AHL signal molecules is shown in FIG. 1, and substances H which appear in the detection of different AHLs signal molecules are detected in two scanning modes 2 N-OH, indicating the presence of AHL signaling molecule, and measuring AHL signaling molecule 3OC at about 18min 8 -HSL。
Example 2:
a method for enhancing microbial degradation of methylene chloride comprising the steps of:
slant culture: inoculating M.rhodesian H13 into an inclined plane LB solid culture medium, and culturing for 30 hours at 30 ℃ to obtain inclined plane thalli;
and (3) performing expansion culture: inoculating the slant thallus obtained in the inoculating loop selecting step into 1L liquid LB culture medium, shake culturing at 30deg.C under 160r/min for 30 hr to obtain OD 600 Bacterial liquid=0.7;
with 1mol/L NaOH aqueous solution or 1mol/L H 2 SO 4 The pH value of the inorganic salt culture medium is regulated to 7.0 by aqueous solution, and the prepared bacterial liquid is inoculated under the condition that the concentration of dichloromethane is 400mg/L, so that the initial bacterial concentration in each parallel sample is OD 600 Calculated as 0.02, 3OC with different contents is exogenously added 8 -HSL (0, 400, 800, 1200, 1600, 2000, 2400 nM). Shaking culture of the sample in shaking table at 30deg.C and 160r/min for 18 hr, sampling, and measuring dichloromethane degradation rate and bacterial liquid OD 600 Value and CO 2 Values, during the course of the experiment, 3 replicates and a blank group without strain were designed. Different 3OC 8 The effect of the exogenous addition of HSL on the dichloromethane degradation (A), growth (B) and mineralization (C) of M.rhodesium H13 is shown in figure 2.
As can be seen from FIG. 2, at 3OC 8 In the range of 800-2400 nM of HSL content, M.rhodesian H13 has higher degradation rate to dichloromethane; in particular 3OC 8 At HSL content=1600 nM, m.rhodesian H13 is optimal (99% or more) for dichloromethane degradation. At the same time, the growth amount of M.rhodesian H13 and the generated CO 2 The values also tended to be consistent with the degradation rate of methylene chloride.
Example 3:
the preparation method of the population induction AHL signal molecule comprises the following steps:
a. slant culture: inoculating M.rhodesian H13 into an inclined plane LB solid culture medium, and culturing for 30 hours at 30 ℃ to obtain inclined plane thalli;
b. and (3) performing expansion culture: inoculating the slant thallus obtained in step a to 1L of liquid LB culture medium by using an inoculating loop, and adding 0.03 g/L4 into the liquid LB culture medium-hydroxy-3-methoxybenzyl alcohol. Shake culturing at 30deg.C and 160r/min for 30 hr to obtain OD 600 Bacterial liquid=0.7;
c. and c, taking the bacterial liquid in the step b, centrifuging at 1000rpm for 15min, taking supernatant, filtering, extracting the filtrate with ethyl acetate in equal volume for three times, combining the extracts, concentrating the nitrogen-blown volatile solvent to 10mL, keeping the AHL signal molecular content at 20mg/L, and keeping the extract at the temperature of-80 ℃ for later use.
Example 4:
simulated exogenous addition of 3OC 8 -the biotrickling tower of HSL handles dichloromethane off-gas:
OD prepared by culturing the method of example 2 600 M.rhodesianum H13 bacterial liquid =0.7 was inoculated into two identical bio-trickling filtration towers (see Zhang Dingfeng, fang Junyi, she Jiexu, etc.. Investigation of bio-trickling filtration towers for purification of multicomponent exhaust gas [ J]Environmental science, 2013.), trickling filtration tower parameters are as follows:
the inoculation amount is 3L, and the spraying amount of the nutrient solution is controlled to be 11 L.h -1 The nutrient solution is replaced every 2d, and is regulated by 0.5mol/L NaOH aqueous solution to maintain the pH value at about 7.0 and the temperature at 30 ℃, and Raschig rings are used as fillers. The residence time in the film forming starting process is 35s, and the inlet concentration of methylene dichloride is 500mg/m 3 . External addition of 160 nM 3OC to the trickling filtration tower 8 -HSL。
Example 5:
external addition of 160 nM 3OC to the trickling filtration tower 8 HSL and 320nM (E) -4-isothiocyanato-1- (methylsulfinyl) -1-butene, the remainder being exactly identical to example 4.
Example 6:
external addition of 160 nM 3OC to the trickling filtration tower 8 HSL and 390nM (E) -4-isothiocyanato-1- (methylsulfinyl) -1-butene, the remainder being exactly identical to example 4.
Example 7:
external addition of 160 nM 3OC to the trickling filtration tower 8 HSL and 300nM (E) -4-isothiocyanato-1- (methylsulfinyl) -1-butene, the remainder being exactly identical to example 4.
Test example 1:
determination of SAM concentration: bacterial solutions of example 1, example 3 were subjected to expansion culture for 8 hours, 12 hours, 20 hours and 30 hours, and were centrifuged at 12000rpm for 5 minutes to obtain bacterial precipitates, and the obtained fermentation cells were subjected to SAM extraction at 4℃for 4 hours or more by mixing 1mL of 1.5M perchloric acid after centrifugation to determine glucose concentration. SAM concentration was determined by HPLC. Chromatographic column: thermo Bio Basic SCX column (4.6 mm. Times.250 mm,5 μm) (Thermo Fisher Scientific, waltham, mass.); mobile phase: 0.1M ammonium formate (pH 4.0 adjusted by formic acid), the flow rate is 1mL min-1; column temperature: 25 ℃; detection wavelength: 254nm. SAM standard series solutions of 5.0, 10.0, 20.0, 40.0, 60.0, 120.0, 240.0mg/L were measured according to the above chromatographic conditions, and the peak area y versus concentration x was used as a standard curve, and the regression equation was: y=44090.957x+566.456, r 2 =0.998. The SAM concentration in the sample is calculated from a SAM sample standard curve of known concentration. The results of the SAM concentration measurement are shown in FIG. 3.
Quantitative detection of ahl signaling molecules: 1L of the bacterial solutions of example 1 and example 3 were subjected to expansion culture for 8h, 12h, 20h and 30h, respectively, were centrifuged at 1000rpm for 15min to obtain supernatants, and the supernatants were filtered, and the filtrates were extracted three times with ethyl acetate in equal volumes, and the extracts were combined and concentrated to 10mL with nitrogen-blown volatile solvent. AHL signal molecules were quantitatively analyzed using a gas chromatograph-mass spectrometer (GC-MS). The gas phase system is HP6890GC, an HP-5MS capillary column (30m*0.25mm ID*0.25um) is equipped, the carrier gas is helium (99.999%), the carrier gas flow rate is 0.8mL/min, the sample inlet temperature is 200 ℃, the split ratio is 150:1, and the auxiliary channel temperature is 280 ℃. Mass spectrometry conditions: ion source (70eV,500uA,MS Quad 150 ℃, MS source 230 ℃). Selecting an ion scan mode: m/z=143. 3OC at 3.0, 5.0, 10.0, 20.0, 30.0, 50.0mg/L 8 The standard series of HSL solutions, determined according to the GC-MS conditions described above, is calibrated by the peak area y versus the concentration x, with the regression equation: y=6515.126x+1944.118, R 2 =0.997. AHL signal molecule-3 OC in sample 8 Concentration of HSL by known concentration 3OC 8 -HSL sample standard curve calculation. 3OC 8 The results of the measurement of the HSL content are shown in FIG. 4.
As can be seen from FIG. 3, the SAM concentration in the bacterial liquid cultured in example 3 is significantly higher than that in example 1; as can be seen from FIG. 4, the culture is performed in example 3Concentrating the cultured fungus liquid to obtain 3OC 8 The HSL content is significantly higher than in example 1, which demonstrates that 4-hydroxy-3-methoxybenzyl alcohol can promote the synthesis of SAM Methylobacterium rhodesianum H, promote the reaction of SAM with acyl donor to homoserine lactone, and promote 3OC 8 Synthesis of HSL such that 3OC 8 The process for the preparation of HSL has the advantage of low cost and high yields.
Test example 2:
measurement of EPS content: the present study represents the EPS content in terms of polysaccharide and protein.
1.1 extraction of EPS: 1) The biofilms in examples 4, 5, 6 and 7 were separated from the filler, placed in a beaker, water was added to the beaker, the pH was adjusted to neutral, and the mixture was stirred uniformly to obtain a suspension sample.
1.2 detection of proteins: 1mL of the suspension sample was taken, and the protein was measured by the biquinolinecarboxylic acid method (Bicinchoninic acid assay, BCA) using SK3501BCA modified kit (Shanghai, china). The method adopts bovine serum albumin as a standard substance, and specific steps refer to a kit instruction.
1.3 detection of polysaccharide: the polysaccharide determination method is a phenol-concentrated sulfuric acid method, uses glucose as a standard substance, and comprises the following specific steps: 1) Adding a small amount of aluminum sheet into phenol, and performing double distillation treatment to remove impurities in the phenol. The treated phenol is prepared into 80% v/v storage solution which is stored in a refrigerator at 4 ℃ in a dark place for standby. The stock solution was diluted to 6% v/v phenol working solution just prior to use (ready to prepare for each assay). 2) 10mg of glucose with constant dry weight is weighed, and distilled water is used for accurately fixing the volume to 100mL to obtain a glucose standard solution with the volume of 100 mug/L. 3) Standard glucose curve drawing: respectively sucking glucose standard solution 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.8 and 1.0mL into test tubes, respectively supplementing distilled water to 2mL, adding 6% phenol 1mL and concentrated sulfuric acid 5mL, standing in boiling water bath for 15min, cooling at room temperature, and standing for 20min. Measuring absorbance at 490nm, drawing a standard curve with polysaccharide concentration as abscissa and absorbance as ordinate, and regression equation of the standard curve is y= 9.2937x-0.0039, R 2 =0.997. 4) 1mL of the sample was taken, 1mL of a 6% phenol solution was added, and then slowly added5mL of concentrated sulfuric acid. Boiling in boiling water bath for 15min, cooling at room temperature, standing for 20min, and measuring absorbance at 490 nm. The concentration value of the actual sample is calculated according to the standard curve. The results of the determination of polysaccharide and protein content are shown in FIG. 5.
2. Determination of biofilm biomass:
setting 3OC without adding exogenous material 8 HSL, remaining conditions and blank control group exactly identical to example 4.
Taking the fillers with biological membranes in examples 4, 5, 6 and 7 and blank control, and leaching the fillers with distilled water; drying the crucible at 105 ℃ in advance, and cooling and weighing the crucible to be W1; placing the filler with the biological film into a crucible, drying for 12 hours at 105 ℃, marking the cooling weight as W2, then placing the crucible into a muffle furnace at 550 ℃ to burn until the weight is constant, marking the cooling weight as W3; soaking the filler in water for 1h, and then stirring with a glass rod to completely remove the biological film; the filler is put into an original crucible, dried for 12 hours at 105 ℃, cooled and weighed to be W4. The calculation formula of the biological membrane biomass W is as follows:
W＝(W2-W3)/(W4-W1)×10 3 mg/g filler
The results of the biological film biological measurement are shown in FIG. 6.
As can be seen from fig. 5, the polysaccharide and protein contents of the biological film of example 5 are significantly higher than those of examples 4, 6 and 7; as can be seen from FIG. 6, the trickling filter packing biomass reached 49mg on day 30 of the film formation in example 4 vss /g Packing material Without exogenously adding 3OC 8 The trickling filter packing biomass of HSL reaches only 28mg vss /g Packing material And example 5 has significantly higher biomass than example 4, example 6, example 7, and examples 4, example 6 are not significantly different and are all significantly higher than example 7, indicating a 3OC 8 The molar ratio of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene to 3OC is 4.2-5.3:1 8 The synergistic effect of HSL can promote cells to secrete EPS, improve the connection effect between bacteria, facilitate the formation of a biological film, increase the amount of the biological film, and ensure that when the addition amount of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene is smaller than the addition amountThe range has no obvious effect, and the range larger than the range has an inhibiting effect.
3. Detection of dehydrogenase Activity: since the dehydrogenase can activate a hydrogen atom of an organic substrate and transfer it to a specific hydrogen acceptor, the dehydrogenase activity may represent biological activity and its ability to metabolize an organic substance.
3.1 preparation of standard curve: taking a2, 3, 5-triphenyltetrazolium chloride (TTC) standard substance, drying the TTC standard substance at 105 ℃ for 2 hours in advance, and preparing TTC standard solutions with the concentrations of 20, 40, 60, 80, 100 and 120 mug/mL. Taking 6 colorimetric tubes, sequentially adding 1mL of TTC standard solution with different concentrations, taking one colorimetric tube without adding TTC standard solution as a control, respectively adding 2mL of Tris-HCl buffer solution and 1mL of 10% Na2S new solution, shaking uniformly, and standing for 20min. After completion of the reaction, 5mL of toluene was added to each tube and shaken well to extract the Triphenylformazan (TPF) thoroughly. And centrifuging at 4000r/min for 10min, taking the upper organic solution to conduct color comparison at the wavelength of 485nm on an ultraviolet spectrophotometer, and drawing a standard curve to obtain a TTC reduced standard curve. y= 0.0322x-0.03355, r 2 ＝0.996。
3.2 preparation of samples: the biofilms of examples 4, 5, 6 and 7 were separated from the filler, placed in a beaker, and water was added to the beaker and stirred well. 1.5mL of the suspension was taken, 2mL of 0.05% TTC was added, and incubated at 37℃for 2h. The resulting TPF was extracted with 6mL of 96% ethanol. The mixture was centrifuged at 3500r/min for 1min, and 1mL of the upper organic solvent was taken and absorbance at 485nm was measured. The results of the measurement were calculated by a standard curve. The test defines the specific activity of dehydrogenase as the amount of TPF measured on 1g dry matter, TPF-specific activity of dehydrogenase (mg TPF/g dry matter). The results of the measurement of the activity of the biofilm dehydrogenase per unit mass are shown in FIG. 7.
As can be seen from FIG. 7, the activity of the biological membrane dehydrogenase per unit mass of example 5 is significantly higher than that of examples 4, 6,
Example 7, example 4, example 6 are significantly higher than example 7, which illustrates a 3OC 8 The molar ratio of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butane to (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butane is in the range of 4.2 to 5.3:1Alkene and 3OC 8 The synergistic effect of HSL can improve the metabolic activity of Methylobacterium rhodesianum H13 cells, and the addition of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene has no obvious effect when the addition amount is smaller than the range, and the inhibition effect when the addition amount is larger than the range.
Test example 3:
a blank control group was set in which no exogenous material was added and the remaining conditions were identical to those of example 4.
Determination of dichloromethane concentration: agilent GC 6890 gas chromatography (GC, HP-Innovax silica gel capillary column, 30m 0.32mm 0.5 μm, J&W Scientific, USA) to quantitatively analyze DCM under the following conditions: the vaporization chamber temperature was 250 ℃, the detector (FID) temperature was 300 ℃, the column temperature was 80 ℃, and the carrier gas flow rate was 1mL min -1 The split ratio is 5:1. The change in concentration of methylene chloride in daily samples was measured to analyze the removal rate of methylene chloride by the strain. The measurement results of the methylene chloride removal rate are shown in FIG. 8.
As can be seen from FIG. 8, the growth of the filler biofilm is consistent with the removal rate results, and the 3OC is externally added in example 4 8 After 15 days of film formation starting, the removal rate of dichloromethane reaches 80.2% -90.1%, and 3OC is not exogenously added 8 The drip filtration tower of HSL has a film formation starting period obviously longer than that of the drip filtration tower, and the methylene dichloride removal rate is only 63.3% after the film formation is started for 30 days; after the film formation in example 5 was started for 10 days, the removal rate of dichloromethane was 82.1% -98.8%, after the film formation in example 6 was started for 17 days, the removal rate of dichloromethane was 80.1% -88.5%, the film formation in example 7 was significantly longer, and after the film formation in example 5 was started for 30 days, the removal rate of dichloromethane was 79.2%, which means that the film formation in example 5 was significantly shorter and the removal rate of dichloromethane was significantly higher, compared with the film formation in example 4, example 6, and example 7, which means that 3OC was 8 The molar ratio of (E) -4-isothiocyanate-1- (methylsulfinyl) -1-butene to 3OC is 4.2-5.3:1 8 The synergistic effect of HSL can reduce the starting time of film formation, strengthen the degradation capability to dichloromethane,the addition amount of (E) -4-isothiocyanato-1- (methylsulfinyl) -1-butene does not have a significant effect when it is smaller than this range, and the inhibition is effected when it is larger than this range.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.

Claims (6)

1. The preparation method of the population induction AHL signal molecule is characterized by comprising the following steps of:
a. slant culture: will beMethylobacterium rhodesianumH13 is inoculated in a slant LB solid culture medium and cultured for 24-36 hours at 30 ℃ to obtain slant thalli;
b. and (3) performing expansion culture: b, inoculating the slant bacterial cells obtained in the step a into an LB liquid culture medium by using an inoculating loop, and culturing for 24-36 h at the temperature of 30 ℃ to obtain OD 600 Bacterial liquid of 0.6-0.8;
c. c, taking the bacterial liquid in the step b, centrifuging at 1000rpm for 15min, taking supernatant, filtering, extracting the filtrate with ethyl acetate in an equal volume for three times, combining the extracts, blowing nitrogen to volatilize the solvent, concentrating, and storing the extract at-80 ℃ for later use;
the final concentration composition of the LB solid medium in the step a is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;
the final concentration composition of the LB liquid medium in the step b is as follows: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
2.A method for enhancing microbial degradation of methylene chloride comprising: the methylene dichloride is taken as a substrate,Methylobacterium rhodesianumh13 is degradation bacteria, inorganic salt culture medium is used as medium, and 3OC is added 8 -HSL degrading dichloromethane at ph=5.5-10, rotation speed 160rpm, temperature 10-40 ℃.
3. The method according to claim 2, characterized in that: the initial concentration of the dichloromethane is 50-1000mg/L,3OC 8 The exogenous addition amount of HSL is 0-2400nM.
4. The method according to claim 2, characterized in that: the 3OC 8 The preparation method of the HSL comprises the following steps: to be used forMethylobacterium rhodesianumH13 is used as a strain to prepare an AHL signal molecule production source, and 3OC is extracted from the AHL signal molecule production source 8 -HSL; the AHL signal molecule production source is OD with inorganic salt culture medium as culture medium 600 0.6 to 0.8Methylobacterium rhodesianumH13 bacterial liquid.
5. The method according to claim 4, wherein: the preparation method of the AHL signal molecule production source specifically comprises the following steps:
1) Slant culture: will beMethylobacterium rhodesianumH13 is inoculated in a slant LB solid culture medium and cultured for 24-36 hours at 30 ℃ to obtain slant thalli;
2) And (3) performing expansion culture: inoculating the slant thallus obtained in the step 1) into LB liquid medium by using an inoculating loop, and culturing for 24-36 h at 30 ℃ to obtain OD 600 Bacterial liquid of 0.6-0.8;
the final concentration composition of the LB solid medium in the step 1) is as follows: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder, 18-20 g/L of agar, deionized water as a solvent and natural pH value;
the final concentration composition of the LB liquid medium in the step 2) is as follows: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
6. The method according to claim 2, characterized in that: the form of the dichloromethane is gas.
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